Novel peptides and combination of peptides for use in immunotherapy against lung cancer, including nsclc, sclc and other cancers

ABSTRACT

The present invention relates to peptides, proteins, nucleic acids and cells for use in immunotherapeutic methods. In particular, the present invention relates to the immunotherapy of cancer. The present invention furthermore relates to tumor-associated T-cell peptide epitopes, alone or in combination with other tumor-associated peptides that can for example serve as active pharmaceutical ingredients of vaccine compositions that stimulate anti-tumor immune responses, or to stimulate T cells ex vivo and transfer into patients. Peptides bound to molecules of the major histocompatibility complex (MHC), or peptides as such, can also be targets of antibodies, soluble T-cell receptors, and other binding molecules.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.16/387,610, filed 18 Apr. 2019, which is a continuation of U.S. patentapplication Ser. No. 16/026,707, filed 3 Jul. 2018, which claimspriority to U.S. Provisional Application No. 62/529,758 filed 7 Jul.2017, and German Patent Application No. 102017115301.2, filed 7 Jul.2017, the content of each is hereby incorporated by reference.

This application is related to PCT/EP2018/067979 filed 3 Jul. 2018, thecontent of which is hereby incorporated by reference.

REFERENCE TO SEQUENCE LISTING SUBMITTED AS A COMPLIANT ASCII TEXT FILE(.txt)

Pursuant to the EFS-Web legal framework and 37 CFR §§ 1.821-825 (seeMPEP § 2442.03(a)), a Sequence Listing in the form of an ASCII-complianttext file (entitled “Sequence_Listing_2912919-089010_ST25.txt” createdon 23 Sep. 2019, and 83,133 bytes in size) is submitted concurrentlywith the instant application, and the entire contents of the SequenceListing are incorporated herein by reference.

BACKGROUND

The present invention relates to peptides, proteins, nucleic acids andcells for use in immunotherapeutic methods. In particular, the presentinvention relates to the immunotherapy of cancer. The present inventionfurthermore relates to tumor-associated T-cell peptide epitopes, aloneor in combination with other tumor-associated peptides that can forexample serve as active pharmaceutical ingredients of vaccinecompositions that stimulate anti-tumor immune responses, or to stimulateT cells ex vivo and transfer into patients. Peptides bound to moleculesof the major histocompatibility complex (MHC), or peptides as such, canalso be targets of antibodies, soluble T-cell receptors, and otherbinding molecules.

The present invention relates to several novel peptide sequences andtheir variants derived from HLA class I molecules of human tumor cellsthat can be used in vaccine compositions for eliciting anti-tumor immuneresponses, or as targets for the development ofpharmaceutically/immunologically active compounds and cells.

DESCRIPTION OF RELATED ART

Lung cancer accounts for the most cancer-related deaths in both men andwomen. Worldwide, lung cancer is the most common cancer in terms of bothincidence and mortality. In 2012, there were more than 1.8 million newcases (13% of total cancer incidence), and 1.6 million deaths (20% oftotal cancer mortality) due to lung cancer. Lung cancer is the leadingcause of cancer death in men in 87 countries and in women in 26countries. More than one third of all newly diagnosed cases occurred inChina. The highest rates are in North America, Europe, and East Asia(World Cancer Report, 2014).

Since 1987, more women have died each year from lung cancer than frombreast cancer. Death rates have continued to decline significantly inmen from 1991-2003 by about 1.9% per year. Female lung cancer deathrates are approaching a plateau after continuously increasing forseveral decades. These trends in lung cancer mortality reflect thedecrease in smoking rates over the past 30 years.

An estimated 230,000 new cases of lung cancer and 160,000 deaths due tolung cancer are expected in 2013 in the USA according to the nationalcancer institute (NCI).

Historically, small cell lung carcinoma (SCLC) has been distinguishedfrom non-small cell lung carcinoma (NSCLC), which includes thehistological types of adenocarcinoma, squamous cell carcinoma, and largecell carcinoma. However, in the past decade, the distinction betweenadenocarcinoma and squamous cell carcinoma has been increasinglyrecognized because of major differences in genetics and also inresponses to specific therapies. Therefore, lung cancers areincreasingly classified according to molecular subtypes, predicated onparticular genetic alterations that drive and maintain lungtumorigenesis (Travis et al., 2013).

Prognosis is generally poor. Of all people with lung cancer, 10-15%survive for five years after diagnosis. Poor survival of lung cancerpatients is due, at least in part, to 80% of patients being diagnosedwith metastatic disease and more than half of patients having distantmetastases (SEER Stat facts, 2014). At presentation, 30-40% of cases ofNSCLC are stage IV, and 60% of SCLC are stage IV.

The 1-year relative survival for lung cancer has slightly increased from35% in 1975-1979 to 44% in 2010, largely due to improvements in surgicaltechniques and combined therapies. However, the 5-year survival rate forall stages combined is only 17%. The survival rate is 54% for casesdetected when the disease is still localized; however, only 16% of lungcancers are diagnosed at this early stage (SEER Stat facts, 2014).

Treatment options are determined by the type (small cell or non-smallcell) and stage of cancer and include surgery, radiation therapy,chemotherapy, and targeted biological therapies such as bevacizumab(AVASTIN®) and erlotinib (TARCEVA®). For localized cancers, surgery isusually the treatment of choice. Recent studies indicate that survivalwith early-stage, non-small cell lung cancer is improved by chemotherapyfollowing surgery. Because the disease has usually spread by the time itis discovered, radiation therapy and chemotherapy are often used,sometimes in combination with surgery. Chemotherapy alone or combinedwith radiation is the usual treatment of choice for small cell lungcancer; on this regimen, a large percentage of patients experienceremission, which is long lasting in some cases surgery (S3-LeitlinieLungenkarzinom, 2011).

Advanced lung cancer has also been resistant to traditionalchemotherapy. However, recent advances have led to exciting progress intherapies that are dependent on histology and genetics. The level ofscrutiny is exemplified by trials of adjuvant chemotherapy designed todifferentiate not only between mutations in codons 12 and 13 of KRAS,but also between different amino acid substitutions as determined byparticular mutations at codon 12 (Shepherd et al., 2013).

To expand the therapeutic options for NSCLC, different immunotherapeuticapproaches have been studied or are still under investigation. Whilevaccination with L-BLP25 or MAGEA3 failed to demonstrate avaccine-mediated survival advantage in NSCLC patients, an allogeneiccell line-derived vaccine showed promising results in clinical studies.Additionally, further vaccination trials targeting gangliosides, theepidermal growth factor receptor and several other antigens arecurrently ongoing. An alternative strategy to enhance the patient'santi-tumor T cell response consists of blocking inhibitory T cellreceptors or their ligands with specific antibodies. The therapeuticpotential of several of these antibodies, including ipilimumab,nivolumab, pembrolizumab, MPDL3280A and MEDI-4736, in NSCLC is currentlyevaluated in clinical trials (Reinmuth et al., 2015).

Considering the severe side-effects and expense associated with treatingcancer, there is a need to identify factors that can be used in thetreatment of cancer in general and lung cancer (including NSCLC andSCLC) in particular. There is also a need to identify factorsrepresenting biomarkers for cancer in general and lung cancer (includingNSCLC and SCLC), leading to better diagnosis of cancer, assessment ofprognosis, and prediction of treatment success.

Immunotherapy of cancer represents an option of specific targeting ofcancer cells while minimizing side effects. Cancer immunotherapy makesuse of the existence of tumor associated antigens.

The current classification of tumor associated antigens (TAAs) comprisesthe following major groups:

a) Cancer-testis antigens: The first TAAs ever identified that can berecognized by T cells belong to this class, which was originally calledcancer-testis (CT) antigens because of the expression of its members inhistologically different human tumors and, among normal tissues, only inspermatocytes/spermatogonia of testis and, occasionally, in placenta.

Since the cells of testis do not express class I and II HLA molecules,these antigens cannot be recognized by T cells in normal tissues and cantherefore be considered as immunologically tumor-specific. Well-knownexamples for CT antigens are the MAGE family members and NY-ESO-1.

b) Differentiation antigens: These TAAs are shared between tumors andthe normal tissue from which the tumor arose. Most of the knowndifferentiation antigens are found in melanomas and normal melanocytes.Many of these melanocyte lineage-related proteins are involved inbiosynthesis of melanin and are therefore not tumor specific butnevertheless are widely used for cancer immunotherapy. Examples include,but are not limited to, tyrosinase and Melan-A/MART-1 for melanoma orPSA for prostate cancer.

c) Over-expressed TAAs: Genes encoding widely expressed TAAs have beendetected in histologically different types of tumors as well as in manynormal tissues, generally with lower expression levels. It is possiblethat many of the epitopes processed and potentially presented by normaltissues are below the threshold level for T-cell recognition, whiletheir over-expression in tumor cells can trigger an anticancer responseby breaking previously established tolerance. Prominent examples forthis class of TAAs are Her-2/neu, survivin, telomerase, or WT1.

d) Tumor-specific antigens: These unique TAAs arise from mutations ofnormal genes (such as β-catenin, CDK4, etc.). Some of these molecularchanges are associated with neoplastic transformation and/orprogression. Tumor-specific antigens are generally able to induce strongimmune responses without bearing the risk for autoimmune reactionsagainst normal tissues. On the other hand, these TAAs are in most casesonly relevant to the exact tumor on which they were identified and areusually not shared between many individual tumors. Tumor-specificity (or-association) of a peptide may also arise if the peptide originates froma tumor- (-associated) exon in case of proteins with tumor-specific(-associated) isoforms.

e) TAAs arising from abnormal post-translational modifications: SuchTAAs may arise from proteins which are neither specific noroverexpressed in tumors but nevertheless become tumor associated byposttranslational processes primarily active in tumors. Examples forthis class arise from altered glycosylation patterns leading to novelepitopes in tumors as for MUC1 or events like protein splicing duringdegradation which may or may not be tumor specific.

f) Oncoviral proteins: These TAAs are viral proteins that may play acritical role in the oncogenic process and, because they are foreign(not of human origin), they can evoke a T-cell response. Examples ofsuch proteins are the human papilloma type 16 virus proteins, E6 and E7,which are expressed in cervical carcinoma.

T-cell based immunotherapy targets peptide epitopes derived fromtumor-associated or tumor-specific proteins, which are presented bymolecules of the major histocompatibility complex (MHC). The antigensthat are recognized by the tumor specific T lymphocytes, that is, theepitopes thereof, can be molecules derived from all protein classes,such as enzymes, receptors, transcription factors, etc. which areexpressed and, as compared to unaltered cells of the same origin,usually up-regulated in cells of the respective tumor.

There are two classes of MHC-molecules, MHC class I and MHC class II.MHC class I molecules are composed of an alpha heavy chain andbeta-2-microglobulin, MHC class II molecules of an alpha and a betachain. Their three-dimensional conformation results in a binding groove,which is used for non-covalent interaction with peptides. MHC class Imolecules can be found on most nucleated cells. They present peptidesthat result from proteolytic cleavage of predominantly endogenousproteins, defective ribosomal products (DRIPs) and larger peptides.However, peptides derived from endosomal compartments or exogenoussources are also frequently found on MHC class I molecules. Thisnon-classical way of class I presentation is referred to ascross-presentation in the literature (Brossart and Bevan, 1997; Rock etal., 1990). MHC class II molecules can be found predominantly onprofessional antigen presenting cells (APCs), and primarily presentpeptides of exogenous or transmembrane proteins that are taken up byAPCs e.g. during endocytosis, and are subsequently processed. Complexesof peptide and MHC class I are recognized by CD8-positive T cellsbearing the appropriate T-cell receptor (TCR), whereas complexes ofpeptide and MHC class II molecules are recognized byCD4-positive-helper-T cells bearing the appropriate TCR. It is wellknown that the TCR, the peptide and the MHC are thereby present in astoichiometric amount of 1:1:1.

CD4-positive helper T cells play an important role in inducing andsustaining effective responses by CD8-positive cytotoxic T cells. Theidentification of CD4-positive T-cell epitopes derived from tumorassociated antigens (TAA) is of great importance for the development ofpharmaceutical products for triggering anti-tumor immune responses(Gnjatic et al., 2003). At the tumor site, T helper cells, support acytotoxic T cell- (CTL-) friendly cytokine milieu (Mortara et al., 2006)and attract effector cells, e.g. CTLs, natural killer (NK) cells,macrophages, and granulocytes (Hwang et al., 2007).

In the absence of inflammation, expression of MHC class II molecules ismainly restricted to cells of the immune system, especially professionalantigen-presenting cells (APC), e.g., monocytes, monocyte-derived cells,macrophages, dendritic cells. In cancer patients, cells of the tumorhave been found to express MHC class II molecules (Dengjel et al.,2006).

Elongated (longer) peptides of the invention can act as MHC class IIactive epitopes. T-helper cells, activated by MHC class II epitopes,play an important role in orchestrating the effector function of CTLs inanti-tumor immunity. T-helper cell epitopes that trigger a T-helper cellresponse of the TH1 type support effector functions of CD8-positivekiller T cells, which include cytotoxic functions directed against tumorcells displaying tumor-associated peptide/MHC complexes on their cellsurfaces. In this way tumor-associated T-helper cell peptide epitopes,alone or in combination with other tumor-associated peptides, can serveas active pharmaceutical ingredients of vaccine compositions thatstimulate anti-tumor immune responses.

It was shown in mammalian animal models, e.g., mice, that even in theabsence of CD8-positive T lymphocytes, CD4-positive T cells aresufficient for inhibiting manifestation of tumors via inhibition ofangiogenesis by secretion of interferon-gamma (IFNγ) (Beatty andPaterson, 2001; Mumberg et al., 1999). There is evidence for CD4 T cellsas direct anti-tumor effectors (Braumuller et al., 2013; Tran et al.,2014).

Since the constitutive expression of HLA class II molecules is usuallylimited to immune cells, the possibility of isolating class II peptidesdirectly from primary tumors was previously not considered possible.However, Dengjel et al. were successful in identifying a number of MHCClass II epitopes directly from tumors (WO 2007/028574, EP 1 760 088B1).

Since both types of response, CD8 and CD4 dependent, contribute jointlyand synergistically to the anti-tumor effect, the identification andcharacterization of tumor-associated antigens recognized by either CD8+T cells (ligand: MHC class I molecule+ peptide epitope) or byCD4-positive T-helper cells (ligand: MHC class II molecule+ peptideepitope) is important in the development of tumor vaccines.

For an MHC class I peptide to trigger (elicit) a cellular immuneresponse, it also must bind to an MHC-molecule. This process isdependent on the allele of the MHC-molecule and specific polymorphismsof the amino acid sequence of the peptide. MHC-class-I-binding peptidesare usually 8-12 amino acid residues in length and usually contain twoconserved residues (“anchors”) in their sequence that interact with thecorresponding binding groove of the MHC-molecule. In this way, each MHCallele has a “binding motif” determining which peptides can bindspecifically to the binding groove.

In the MHC class I dependent immune reaction, peptides not only have tobe able to bind to certain MHC class I molecules expressed by tumorcells, they subsequently also have to be recognized by T cells bearingspecific T cell receptors (TCR).

For proteins to be recognized by T-lymphocytes as tumor-specific or-associated antigens, and to be used in a therapy, prerequisites must befulfilled. The antigen should be expressed mainly by tumor cells andnot, or in comparably small amounts, by normal healthy tissues. In apreferred embodiment, the peptide should be over-presented by tumorcells as compared to normal healthy tissues. It is furthermore desirablethat the respective antigen is not only present in a type of tumor, butalso in high concentrations (i.e. copy numbers of the respective peptideper cell). Tumor-specific and tumor-associated antigens are oftenderived from proteins directly involved in transformation of a normalcell to a tumor cell due to their function, e.g. in cell cycle controlor suppression of apoptosis. Additionally, downstream targets of theproteins directly causative for a transformation may be up-regulated undthus may be indirectly tumor-associated. Such indirect tumor-associatedantigens may also be targets of a vaccination approach (Singh-Jasuja etal., 2004). It is essential that epitopes are present in the amino acidsequence of the antigen, in order to ensure that such a peptide(“immunogenic peptide”), being derived from a tumor associated antigen,leads to an in vitro or in vivo T-cell-response.

Basically, any peptide able to bind an MHC molecule may function as aT-cell epitope. A prerequisite for the induction of an in vitro or invivo T-cell-response is the presence of a T cell having a correspondingTCR and the absence of immunological tolerance for this particularepitope.

Therefore, TAAs are a starting point for the development of a T cellbased therapy including but not limited to tumor vaccines. The methodsfor identifying and characterizing the TAAs are usually based on the useof T-cells that can be isolated from patients or healthy subjects, orthey are based on the generation of differential transcription profilesor differential peptide expression patterns between tumors and normaltissues. However, the identification of genes over-expressed in tumortissues or human tumor cell lines, or selectively expressed in suchtissues or cell lines, does not provide precise information as to theuse of the antigens being transcribed from these genes in an immunetherapy. This is because only an individual subpopulation of epitopes ofthese antigens are suitable for such an application since a T cell witha corresponding TCR has to be present and the immunological tolerancefor this particular epitope needs to be absent or minimal. In a verypreferred embodiment of the invention it is therefore important toselect only those over- or selectively presented peptides against whicha functional and/or a proliferating T cell can be found. Such afunctional T cell is defined as a T cell, which upon stimulation with aspecific antigen can be clonally expanded and is able to executeeffector functions (“effector T cell”).

In case of targeting peptide-MHC by specific TCRs (e.g. soluble TCRs)and antibodies or other binding molecules (scaffolds) according to theinvention, the immunogenicity of the underlying peptides is secondary.In these cases, the presentation is the determining factor.

SUMMARY

In a first aspect of the present invention, the present inventionrelates to a peptide comprising an amino acid sequence selected from thegroup consisting of SEQ ID NO: 1 to SEQ ID NO: 489 or a variant sequencethereof which is at least 77%, preferably at least 88%, homologous(preferably at least 77% or at least 88% identical) to SEQ ID NO: 1 toSEQ ID NO: 489, wherein said variant binds to MHC and/or induces T cellscross-reacting with said peptide, or a pharmaceutical acceptable saltthereof, wherein said peptide is not the underlying full-lengthpolypeptide.

The present invention further relates to a peptide of the presentinvention comprising a sequence that is selected from the groupconsisting of SEQ ID NO: 1 to SEQ ID NO: 489 or a variant thereof, whichis at least 77%, preferably at least 88%, homologous (preferably atleast 77% or at least 88% identical) to SEQ ID NO: 1 to SEQ ID NO: 489,wherein said peptide or variant thereof has an overall length of between8 and 100, preferably between 8 and 30, and most preferred of between 8and 14 amino acids.

The following tables show the peptides according to the presentinvention, their respective SEQ ID NOs, and the prospective source(underlying) genes for these peptides. In Table 1, peptides with SEQ IDNO: 1 to SEQ ID NO: 83 bind to HLA-A*24, peptides with SEQ ID NO: 84 toSEQ ID NO: 133 bind to HLA-A*02, peptides with SEQ ID NO: 134 to SEQ IDNO: 201 bind to HLA-A*01, peptides with SEQ ID NO: 202 to SEQ ID NO: 219bind to HLA-A*03, peptides with SEQ ID NO: 220 to SEQ ID NO: 295 bind toHLA-B*07, peptides with SEQ ID NO: 296 to SEQ ID NO: 318 bind toHLA-B*08, peptides with SEQ ID NO: 319 to SEQ ID NO: 374 bind toHLA-B*44. The peptides in Table 2 have been disclosed before in largelistings as results of high-throughput screenings with high error ratesor calculated using algorithms, but have not been associated with cancerat all before. In Table 2, peptides with SEQ ID NO: 375 to SEQ ID NO:387 bind to HLA-A*24, peptides with SEQ ID NO: 388 to SEQ ID NO: 393bind to HLA-A*02, peptides with SEQ ID NO: 394 to SEQ ID NO: 452 bind toHLA-A*01, peptides with SEQ ID NO: 453 to SEQ ID NO: 458 bind toHLA-A*03, peptides with SEQ ID NO: 459 to SEQ ID NO: 475 bind toHLA-B*07, peptides with SEQ ID NO: 476 to SEQ ID NO: 489 bind toHLA-B*44. The peptides in Table 3 are additional peptides that may beuseful in combination with the other peptides of the invention. In Table3, peptides with SEQ ID NO: 490 to SEQ ID NO: 508 bind to HLA-A*24,peptides with SEQ ID NO: 509 to SEQ ID NO: 528 bind to HLA-A*02,peptides with SEQ ID NO: 529 to SEQ ID NO: 530 bind to HLA-B*07, peptidewith SEQ ID NO: 531 binds to HLA-B*44.

TABLE 1 Peptides according to the present invention. Seq ID HLA NoSequence Gene(s) allotype   1 QYDPTPLTW ADAMTS12 A*24   2 VWSNVTPLKFMMP12 A*24   3 YLEKFYGL MMP12 A*24   4 SYEKVINYL MAGEA9, MAGEA9B A*24  5 RYMKKDYLI SLC35D3 A*24   6 KYKDYFPVI MAGEC2, LOC392555 A*24   7VQQWSVAVF PTHLH A*24/B*15   8 PFLPPAACFF ASCL1 A*24   9 RILRFPWQL MMP11A*24/A*32  10 VWSDVTPLNF MMP13 A*24  11 YYSKSVGFMQW FAM111B A*24  12STIRGELFFF MMP11 A*24/B*57  13 HYTYILEVF SLC7A11 A*24  14 SYSSCYSFKRT13, KRT17 A*24  15 KYALLLQDL PLEKHG4B A*24  16 TYNPDFSSL SP9 A*24  17YYADKKTFIVL SCN9A A*24  18 DYIGSVEKW HS6ST2 A*24  19 ILKEDPFLF MACC1A*24  20 EFTTVLYNF TP63 A*24  21 SYEVRSTF TAS2R38 A*24  22 TQPGDWTLFPOSTN A*24/B*15  23 KFIISDWRF FAM83A A*24  24 MYPDLSELLM NUP155 A*24  25SYNGYVFYL ROS1 A*24  26 KTPTNYYLF NMUR2 A*24/B*35  27 NYTLYPITF GXYLT1A*24  28 YYSIISHTL ROS1 A*24  29 VYPLLSRLYW ROS1 A*24  30 QYLPGWTVLFSLC22A31 A*24  31 QYQNVLTLW DST A*24  32 SLPDLTPTF LAMB3 A*24  33KSSVIASLLF TMTC3 A*24/B*57  34 MQPRMFFLF FGD6 A*24  35 KYLEESVWL GPR98A*24  36 KQMEDGHTLF UHRF1 A*24/B*15  37 QWPWQASLQF TMPRSS3 A*24  38KYTNWKAFL MBTD1 A*24  39 LIFMLANVF GABRP A*24/A*32  40 QYEPPSAPSTTFDROSHA A*24  41 VIYFMGAIF OR7E24 A*24/B*15  42 TLPNTIYRF ROS1 A*24  43IQMDEPMAF CDC7 A*24/B*15  44 AYLSAVGTF ABCC1 A*24  45 KYFVPPQLF B3GNT6A*24  46 AFPVTSIFHTF KCNG1, KCNG2 A*24  47 KYADYFLEV CCNJ A*24  48VFIDHPVHLKF TENM4 A*24  49 LYISEVRNI DST A*24  50 SYPELVKMVW C5orf34A*24  51 KYALLLQEL PLEKHG4 A*24  52 KYMKIFHKF ZNF681 A*24  53 KYITNLEDLTXNDC16 A*24  54 LLIKLLQTF PRKDC A*24/B*15  55 RWMDQRLVF GABRP A*24  56VYMIEPLEL ADAM23 A*24  57 YPSIIQEF POLA1 A*24  58 QFAAPLRGIYF C1QTNF6A*24  59 KYSTTFFMV XPR1 A*24  60 TYLSIFDQL SF3A3 A*24  61 NYAENILTLFIGNL1 A*24  62 LYQEILAQL URB1 A*24  63 VMPSDSFFF GAL3ST4 A*24  64NYAIFDEGHML SMARCAD1 A*24  65 VYPASKMFPFI CKAP5 A*24  66 IYFRDSSFL TEP1A*24  67 RYPGKFYRV ZAK A*24  68 IYQQIIQTY NCAPG2 A*24  69 IMPEKFEFW CHD2A*24  70 PYTNYTFDF CNOT6, CNOT6L, A*24 CNOT6LP1  71 SYMVLAPVFSPIN1, SPNS1 A*24  72 RYEGILYTI LSM14A, LSM14B A*24  73 SYIGLPLTL NPC1A*24  74 VYDQYFITL ATP8B2 A*24  75 NYIYSISVF PLAA A*24  76 WYGWHFPELNOP58 A*24  77 AYTLLGHEFV CDC27 A*24  78 TWFPKTPMLF KBTBD2 A*24  79RYLADLPTL CEP85 A*24  80 YYSPLRDLL Rasa3 A*24  81 LYPEGLRLL ATP6V0D2A*24  82 RFLPSPVVI CUL3 A*24  83 TYCQNIKEF EIF3H A*24  84 YVDINTFRLMMP12 A*02  85 YIDEFQSLV RTL1 A*02  86 FVIDGFDEL NLRP2 A*02  87TLYPYQISQL KIF26B A*02  88 VQMVITEAQKV LAMC2 A*02  89 ILSTTMVTV KIF26BA*02  90 FLLMHPSI NDST4 A*02  91 FALPGLLHA LAMB3 A*02  92 NLRDLLSEVKIF26B A*02  93 TLQEKILQV MYO3A A*02  94 VLPDIETLIGV TET1 A*02  95ITIGVLARV CEACAM6 A*02  96 HLVGGLHTV DACH1, DACH2 A*02  97 VLALVNSTVCBFA2T2 A*02  98 LQSSGLTLLL MSLNL A*02/B*13  99 FLKEKVPGI CDKAL1 A*02100 RQYPTPFQL ZNF280C A*02/B*48 101 FIISDWRFVL FAM83A A*02 102 SLLEQAIALST18 A*02 103 FLYYPDPVL PLXNA1 A*02 104 GMLDIFWGV RASSF6 A*02 105SLLTHIPTA PLEKHG4 A*02 106 FIIDTTYPAYV FAP A*02 107 LLQGAIESV PLEKHG4A*02 108 MIIALSLYI GPR33 A*02 109 LLLGSIGLLGV OPN3 A*02 110 LLADFQALLCCDC87 A*02 111 ALCLLLHLL MRGPRE A*02 112 SVSDGIHSV CELSR1 A*02 113AVLTGLVEV LRBA A*02 114 ILDERQVLL ITGAE A*02 115 MLLETQDALYV ADORA2BA*02 116 VLMEENSKL HAP1 A*02 117 FLDPNARPLV CAD A*02 118 ALSSVLHSI FGD6A*02 119 RTADITVTV ITGAE A*02 120 ALLANLPAV SLC26A9 A*02 121 ALVDTLTGIIPO9 A*02 122 ALLEMFPEITV TXNDC16 A*02 123 LMAFFLAVV OR6C75 A*02 124SVASVLLYL PRKDC A*02 125 VLQPFLPSI IPO9 A*02 126 FLSTVTSV SOGA2 A*02 127GLDGSLVFL MARCH6 A*02 128 FLGTTPTL SF3B3 A*02 129 VLYDKDAVYV BMS1 A*02130 NLWGGQGLLGV GORASP2 A*02 131 LLKEFVQRV COL6A3 A*02 132 ALWLVDPLTVSLC33A1 A*02 133 MTLPVDAVISV UFSP2 A*02 134 AAEIGDKSWLY PLA2G7 A*01 135ASEDSVLLY CHAF1B A*01 136 ATDLVVLDRY DICER1 A*01 137 ATSKFMEFY EDNRAA*01 138 DSDSCHFNY DCBLD2 A*01 139 ECDMAFHIY UHRF1, LOC728688 A*01 140ESDREELNY CBFA2T2 A*01 141 ESDVGVVVY DDX60L A*01 142 EVAEPSVLFDLYC9orf114 A*01 143 FIDYPKKEDY PLAU A*01 144 FLDSQNLSAY BBS1, DPP3 A*01145 FVDKPVAY TAF1B A*01 146 GLNTGSALSY COL6A3 A*01/B*15 147 GSSDSSTLPKLTDRD5 A*01 148 GTEFTTILY TP73 A*01 149 GTEFTTVLY TP63 A*01 150 GTELLSLVYPRKDC A*01 151 HSDLKVGEY DICER1 A*01 152 HTDSLHLLI ANKRD52, FLJ25613A*01 153 KLDRSVFTAY FAM111B A*01 154 LLDISQKNLY ZNF439 A*01 155LLDPNPHMY RGS17 A*01 156 LLDSLREQY SESTD1 A*01 157 LMDRPIFY GALNS A*01158 LSDLLKQGY PKD2L1 A*01 159 LSDTSVIQFY C16orf62 A*01 160 LTEAVLNRYKIF26B A*01 161 LVDDGTHGQY TRPM2 A*01 162 LVDNSIRELQY CARD8 A*01 163NSDSSLTLREFY FSTL4 A*01 164 NTDNNLAVY CXorf61 A*01 165 NTDPTAPPY CDH3A*01 166 NTQITDIGRY HMCN1 A*01 167 QSDPGTSVLGY SEZ6 A*01 168QTDHPQPILDRY ITGAE A*01 169 RLDTPLYFSY LEPRE1 A*01 170 RSDDTAVYYIGHA1, IGHA2, A*01 IGHD, IGHG3, IGHV1-18, IGHV1-2, IGHV4-31 171RSDPVTLNVLY CEACAM6, PSG1, A*01 PSG2, PSG4, PSG5, PSG7, PSG8 172RTDSCSSAQAQY DCBLD2 A*01 173 RTEFNLNQY COL12A1 A*01 174 SADDIRGIQSLYMMP12 A*01 175 SDVTPLTF MMP11 A*01 176 SRTINVSNLY LAMA1 A*01 177SSDEVNFLVY TBL1XR1 A*01 178 SSDSSTLPKL TDRD5 A*01/C*12 179 STAKSATWTYTP63, TP73 A*01 180 STDPWIQMAY KDM1B A*01 181 TADGKTYYY TCERG1 A*01 182TDYHVRVY FNDC3B A*01 183 TLEDIATSHLY CBFA2T2 A*01 184 TSAHPEDSSFYLOC731467, A*01 TRBV20-1 185 TSDSNLNKY KCNH7 A*01 186 TTDIIEKY DDX60LA*01 187 VADLHLYLY GARS A*01 188 VSDAKLDKY RCOR2, LOC441644 A*01 189VSDSECLSRY LAMA1 A*01 190 VTDGINPLIDRY FREM2 A*01 191 VTDGSLYEGVAY DSEA*01 192 VTEESFDSKFY CDKAL1 A*01 193 VTEFSLNTY COL6A3 A*01 194VVADTKMIEY ADAMTS12 A*01 195 VVDSVGGYLY ROS1 A*01 196 WMFFVINY TMEM217A*01 197 YADTVRPEFY COL6A3 A*01 198 YLDPVQRDLY ZNF655 A*01 199YLPQHTIETY TP63 A*01/B*15 200 YSDEDVTKY SDK2 A*01 201 YVGKEHMFYMAGEA9, MAGEA9B A*01 202 KLAELEGALQK KRT81, KRT121P, A*03 KRT83, KRT85,KRT86 203 KVKDTPGLGK KIF26B A*03 204 AVFDKFIRY BTBD17 A*03 205 SLDGAARPKSP6 A*03 206 KLIDLSQVMY MACC1 A*03/B*15 207 RSFNGLLTMY LAMB3 A*03/B*15208 GLASRILDAK LAMB3 A*03 209 RTQIPMSEK RASSF6 A*03 210 ATSGVPVYKSLC44A5 A*03 211 TVNPVAIHK GLI2 A*03 212 KAYEQVMHY FOXA2 A*03 213LNINMTSPMGTK PCSK2 A*03 214 RTMSEAALVRK RASSF6 A*03 215 MMFSGPQILKLABCC1 A*03/A*32 216 KLYAWELAF ABCC1 A*03/A*32 217 RILNQILYY FGD6 A*03218 KTLVAELLILK POLQ A*03 219 RLRSSLVFK FAM83B A*03 220 SPSVSQLSVL PRAMEB*07 221 VPDVAQFVL MMP1 B*07 222 NPFYPEVEL MMP1 B*07 223 YPKDIYSSF MMP1B*07 224 GPQPWHAAL MMP11 B*07 225 LPFDGPGGIL MMP11 B*07 226 SPRMSGLLSQTDLL3 B*07 227 YPRGNHWAVGH GRP B*07 228 YPRGNHWAVGHL GRP B*07 229VPLPAGGGTV GRP B*07 230 VPLPAGGGTVL GRP B*07 231 RPRALRDLQL NLRP7 B*07232 RPRALRDLQLL NLRP7 B*07 233 KPYQGNPTF DNAH17 B*07 234 RAKNAGVTI LAMC2B*07 235 MPLKHYLLL LRRC15 B*07 236 RVRGGEDGDRAL INSM1 B*07 237RPAATAVISL SLC7A11 B*07 238 KPGPPWAAF DCBLD2 B*07 239 YVPSASLFML E2F7B*07 240 SPREVTTVL DCBLD2 B*07 241 SARLATDAL FAM83A B*07 242 SPRWLPVSLBTBD17 B*07 243 RPIENRILIL PSG1, PSG3, PSG4, B*07 PSG5, PSG6, PSG7,PSG8, PSG9 244 FPYVRDFVM COL6A3 B*07/B*35 245 RIREHVPQL COL6A3 B*07 246TPLPAVIVL SLC7A11 B*07 247 RALLARLLL PLAU B*07 248 IPNWARQDL NLRP7 B*07249 VPSSRILQL THEG B*07 250 SPRDFLSGL ABCA2, TPH1 B*07 251 VPRSSGQTV SP6B*07 252 SPDIRNTTV DCBLD2 B*07 253 RVIDAVRFTL TP63 B*07 254 NPFPHLITLROS1 B*07 255 MPLLENLYL MXRA5 B*07 256 SPRVPSIEL COL7A1 B*07 257LPRIPFADV ROS1 B*07 258 LPRGPLASL CDH3 B*07 259 RPPAAGLRGISL SEZ6L B*07260 YPQHPGLNA SOX2 B*07 261 APSARVGVC KRT86 B*07 262 SAYPQRLEI CYP24A1B*07/B*51 263 HPAPYGDLL GLI2 B*07 264 RPILIIITL TP73 B*07 265 SPRQPPRLVCYP24A1 B*07 266 HAYPPGPGL MMP10 B*07/C*03 267 HPELVNHIVF GALNT5B*07/B*35 268 YPLFRGINL COL5A1 B*07 269 APRAPRLML ITGA3 B*07 270APGPRFLVT CD109 B*07 271 MPLPWSLALP EGFL6 B*07/B*35 272 MPLPWSLALPLEGFL6 B*07 273 MPLLWLRGF INHBA B*07/B*35 274 TPYQEHVAL ZNF618 B*07/B*35275 APHPPLSVV IQGAP3 B*07 276 LPRAGGAFL LAMB3 B*07 277 MPLFEPRVF PTK7B*07/B*35 278 HPMIDINGIIVF COL5A1 B*07/B*35 279 SPARASPAL TMPRSS13 B*07280 VPISEEGTPVL KIAA0754 B*07 281 RPRAPVTPA HES6 B*07 282 MPQIETRVILECT2 B*07 283 RPHSLSSEL ITGAE B*07 284 FPVTSIFHTF KCNG1, KCNG2 B*07/B*35285 FPSFLTNSL CEP192 B*07/B*35 286 VPTLRSEL DST, MACF1 B*07 287APREEQQRSL KIAA1211 B*07 288 FPQKFIDLL SASS6 B*07 289 VPENHSVAL FAM83BB*07 290 APYRPPDISL TANC2 B*07 291 SPQRLRGLL CTHRC1 B*07 292 SPQRLRGLLLCTHRC1 B*07 293 RPRSALPRLLLP FZD2 B*07 294 GPTPNTGAAL COL6A3 B*07 295KPEGTRIAV COL6A3 B*07 296 MPMQDIKM PRAME B*08 297 RAQLKLVAL KLHDC7B B*08298 FNKRKPLSL NLRP2 B*08 299 MAQFKEISL NLRP2 B*08 300 VASPKHCVL KIF26BB*08 301 YMHKLLVL PTH2 B*08/B*35 302 HLLQKQTSI TP63 B*08 303 LPFPKFTVGALNT5 B*08 304 ELKKLYCQI TP63 B*08 305 ALKLRVAVL C16orf59 B*08 306ILKVKVGL POSTN B*08 307 ILLPRTVSL MXRA5 B*08 308 MLKQKVEEL DST B*08 309DAIQRKYSC DST B*08 310 LPPKKFVL NUP155 B*08 311 EIRIRVVQM PRKDC B*08 312EAMLRNKEL CENPF B*08 313 ELKKKEYEEL CENPF B*08 314 AIISRLVAL TANC2 B*08315 DIYQRALNL VPS13B B*08 316 VIKEKALTL USP9X, USP9Y B*08 317 LVKVKVLLARID4A B*08 318 EAAIRSVEL DSCR3 B*08 319 AEMLERVIKNY MAGEA4 B*44 320MEVDPIGHVYIF MAGEA3, MAGEA6 B*44 321 AEMLESVIKNY MAGEA1, MAGEA8, B*44MAGEA9, MAGEA9B 322 KEVDPAGHSY MAGEA8, MAGEA9, B*44 MAGEA9B 323 SEFMQVIFMAGEA9, MAGEA9B B*44 324 TDSIHAWTF SLC35D3 B*44/B*18 325 QEQDVDLVQKYMMP1 B*44 326 QEMQHFLGL MMP12 B*44 327 YEIEARNQVF MMP12 B*44/B*18 328FEYDFLLQRI MMP12 B*44 329 NEHPSNNW LAMC2 B*44 330 KEGDLGGKQW ADAMTS12B*44 331 EDAQGHIW MMP11 B*44 332 MEVPVIKI ECT2 B*44 333 AETLSTIQI KIF26BB*44 334 AEDEPAAAHL KIF26B B*44 335 KELEATKQY KIF26B B*44 336 ASSSGPMRWWLAMB3 B*44/B*57 337 TENRYCVQL JUP, KRT13, KRT17 B*44 338 SEGSEPALLHSWFAM83A B*44 339 SEPALLHSW FAM83A B*44 340 TEFSLNTY COL6A3 B*44 341EEIEGKGSFTYF POSTN B*44 342 HEFSSPSHL TP63 B*44 343 TEFTTVLY TP63 B*44344 EEATGQFHVY CEACAM 1, CEACAM3, B*44 CEACAM6 345 IEFIHPQAF MXRA5 B*44346 VEAPGPVHVYW PTK7 B*44 347 ALNPYQYQY DLX5 B*44/A*29 348 AEIQGNINHVIQGAP3 B*44 349 AEQDMRELTY DST B*44 350 GECDVFKEIL DCBLD2 B*44 351EEVNYINTF CCNE2 B*44 352 NEVLTYIKF ABCC5 B*44 353 GEIIMQNNW SERTAD4 B*44354 TEDPTILRI PLXNA1 B*44 355 SDMVRFHLF SGK196 B*44 356 EEGRVYLF ITGA2B*44 357 RELENCFQIQ DNAH14 B*44 358 KEADIHFLI COL6A5 B*44 359 DELFSIALYNUP155 B*44 360 AEVPTGVII ITGA2 B*44 361 SENLFFASF ITGA2 B*44 362SEKGVIQVY NUP155 B*44 363 AELDKLTSV CENPF B*44 364 AETPIQNVI MET B*44365 SEMNVNMKY MET B*44 366 AENLFRAF PRKDC B*44 367 GEVHPSEMI PRKDC B*44368 GEFPVRVQV YEATS2 B*44 369 EEIERFFKL NUP155 B*44 370 YEDLSQKY CENPFB*44 371 GELALKKKI PRKDC B*44 372 TEGIIMKDF MET B*44 373 MEMQKSPVFFSTL4, GRM7, B*44 LOC440173, LOC644919, LOC728755, SLC44A5 374 DEVNFLVYTBL1X, TBL1XR1, B*44/B*18 TBL1Y

TABLE 2 Additional peptides according to thepresent invention with no prior known cancer association. Seq HLA ID NoSequence Gene(s) allotype 375 VYSDLHAFYY MANEAL A*24 376 KYVKDFHKFZNF724P A*24 377 VYVGAVNRI PLXNA1 A*24 378 KFLGPAEHLTF PROM2 A*24 379NYIVPDKQIF POLA1 A*24 380 VFQEKHHVI MOXD1 A*24 381 TYSKKHFRI CHEK2 A*24382 IYHSHHPTL OPA1 A*24 383 RYKQDVERF SMC5 A*24 384 KYVKVFDKF ZNF107A*24 385 MYINEVERL PTPN14 A*24 386 VYNDHSIYVW MAPKBP1 A*24 387RWLPQKNAAQF DOCK5, PPP2R2A A*24 388 FSIPEGALVAV ABCC1 A*02 389TLMEQPLTTL TXNDC16 A*02 390 HIMPTVHTV ADNP2 A*02 391 SLIDMRGIETV SMC6A*02 392 SLFKDQMEL IPO8 A*02 393 ILLPYLQTL TIPARP A*02 394 ASEAEMRLFYDHX37 A*01 395 ASEASRLAHY HIST1H2BA, HIST1H2BL, A*01 HIST3H2BB 396ASEFGNHYLY SF3B3 A*01 397 ASEITSKGASLY CLUAP1 A*01 398 ASEQQALHTVQYNUP188 A*01 399 ATDIPCLLY RINT1 A*01 400 ATDISRQNEY PDE7A A*01 401DSDESYMEKSLY CLSPN A*01 402 DTDSQRLAY E2F1 A*01 403 ELDSKVEVLTY SNX7A*01 404 ETARKFLYY GPD2 A*01 405 ETEEGIYWRY KREMEN2 A*01 406 ETEQTKFWDYFUT11 A*01 407 FSDNDKLYLY RFX5 A*01 408 FTEQWTDGY TLK2 A*01 409FVDPLVTNY TRIT1 A*01 410 GSDHQSPSSSSY ZBTB43 A*01 411 GTVYEDLRY UBE2CA*01 412 ILDEVIMGY KIF11 A*01 413 ISDRYYTALY CEBPZ A*01 414 KTDESLTKYCHD8 A*01 415 LLDPRSYHTY DOCK8 A*01 416 LLDTAQKNLY ZNF614 A*01 417LLEDKHFQSY WDR6 A*01 418 LSDPSGPKSY RPS6KC1 A*01 419 LSELKPMSY TMTC3A*01 420 LTEDKETLQY TUBGCP2 A*01 421 LTELLERAAFY SLC15A4 A*01 422MIDVTKSYY DCTN5 A*01 423 NLDAVHDITVAY LCLAT1 A*01 424 NLDEEKQLLY FRMD6A*01 425 NLDIIQQEY WDR75 A*01 426 NLDQATRVAY SMC4 A*01 427 NSDEQKITEMVYLRBA A*01 428 NSELSCQLY TYMS A*01 429 NTEDSSMSGYLY FGD6 A*01 430NTEGLHHLY SMEK2, SMEK3P A*01 431 NTSDMMGRMSY ARID1A A*01 432 NVDPVQHTYAGRN A*01 433 QIDTGENLY ZNF267 A*01 434 QTDCAPNNGY NOMO1, NOMO2, NOMO3A*01 435 QTDDTWRTEY ZMYM2 A*01 436 QTETGTPYMLY RRM1 A*01 437 STDGKHVWVEYCCNT1, CCNT2 A*01 438 STDNFNCKY FGD6 A*01 439 TLDAGKFQIY DHX15 A*01 440TLDENPGVRY STXBP3 A*01 441 TLDSALNAASYY TCTN3 A*01 442 TSDFSRFTNY CCNE2A*01 443 TTDFPSESSFEY CXorf21 A*01 444 TTDTVIRSY SETD4 A*01 445VLDQGKITEY ABCB10 A*01 446 VTAQVVGTERY PLD2 A*01 447 VVDEDHELIY CHST11A*01 448 YLDIPNPRY CIT A*01 449 YLDRGTGNVSFY TRIM4 A*01 450 YSDDGQKWTVYDCBLD2 A*01 451 YSDSLVQKGY MSH6 A*01 452 YVDAVLGKGHQY NUP160 A*01 453AINTSIKNK TRPM8 A*03 454 KVYTPSISK CDKAL1 A*03 455 RIADIFVKK FGD6 A*03456 SMFTAILKK LRBA A*03 457 SINKPTSER NDC80 A*03 458 GIADFVLKY RNFT2A*03 459 RPMQQARAQL KLHDC7B B*07 460 MPMAGDMNGL TP63 B*07 461 RPILIIVTLTP63 B*07 462 RPFHTRATV KIF26B B*07 463 TPKAGPTL KIF26B B*07 464YPRPGTPAA CDKAL1 B*07 465 VPRPIFSQL GREB1, GREB1L B*07 466 APYKSVTSLFGD6 B*07 467 KPFSSFTSM RASSF6 B*07 468 SPMYGQAGL FOXA2 B*07 469YPENGVVQM UHRF1 B*07 470 SPNSYFRVL PCDHB13, PCDHB8 B*07 471 KPRPDVTNELCDCA7 B*07 472 NPRATDAQL LRBA B*07 473 LPRALLSSL IL4I1 B*07 474 LPRLLPALHEATR2 B*07 475 RPHKPGLYL MANEA B*07 476 AEEEIMKKI IGF2BP3 B*44 477QENSYQSRL LAMC2 B*44 478 SEIEQEIGSL LAMC2 B*44 479 AEIQPQTQV PTK7 B*44480 GEVSGLTKDF CDKAL1 B*44 481 RELQHEHSL FAT1 B*44 482 TEREWADEW CBFA2T2B*44 483 EENDQSTHKW YEATS2 B*44 484 AEVGFVRFF MSH2 B*44 485 SEIEDSTKQVFBRCA2 B*44 486 SEDDPILQI NUP155 B*44 487 AEDQLHHSF GXYLT1 B*44 488TEFPIIKMY TXNDC16 B*44 489 SEIGKAVGF CLSPN B*44

TABLE 3 Peptides according to the presentinvention useful for e.g. personalized cancer therapies. Seq HLA ID NoSequence Gene(s) allotype 490 SYVKVLHHL MAGEA12, A*24 LOC101060230 491KYLEKYYNL MMP1 A*24 492 NYEDHFPLL MAGEA10 A*24 493 TYKYVDINTF MMP12 A*24494 RYLEKFYGL MMP12 A*24 495 SYNDALLTF TRPM8 A*24 496 VFMKDGFFYF MMP1A*24 497 NYPKSIHSF MMP12 A*24 498 EYIRALQQL ASCL1 A*24 499 VYFVAPAKFLAMC2 A*24 500 VWSDVTPLTF MMP11 A*24 501 GYIDNVTLI LAMC2 A*24 502SVHKITSTF LAMC2 A*24 503 VHFEDTGKTLLF MMP13 A*24 504 VYEKNGYIYF MMP13A*24 505 AYISGLDVF DNAH17 A*24 506 RYVFPLPYL SOX14 A*24 507 VYIAELEKISMC1B A*24 508 IYVTGGHLF KLHDC7B A*24 509 ALLEEEEGV MAGEA4 A*02 510KVLEHVVRV MAGEA4, MAGEA8 A*02 511 KIWEELSVLEV MAGEA3, MAGEA6 A*02 512VLGEEQEGV MAGEA9, MAGEA9B A*02 513 KLVELEHTL CXorf61 A*02 514VQLDSIEDLEV PRAME A*02 515 KIFEMLEGV CT45A1, CT45A2, A*02CT45A3, CT45A4, CT45A5, CT45A6, LOC101060208, LOC101060210, LOC101060211516 YTFSGDVQL MMP1 A*02 517 TLYNPERTITV IGF2BP1, IGF2BP3 A*02 518GLLEDERALQL MEX3A A*02 519 KIQEILTQV IGF2BP3 A*02 520 KIQEMQHFL MMP12A*02 521 FVYGEPREL MAGEC2, LOC392555 A*02 522 TLDEKVAEL MAGEC2 A*02 523HLIAEIHTA PTHLH A*02 524 KVWSDVTPL MMP11, MMP13 A*02 525 RLDDLKMTV LAMC2A*02 526 VLSPFILTL KLHDC7B A*02 527 LLDSVSRL LAMC2 A*02 528 RLLDSVSRLLAMC2 A*02 529 HPSAHDVIL LAMC2 B*07 530 APAAWLRSAA MMP11 B*07 531AEIEADRSY LAMC2 B*44

The present invention furthermore generally relates to the peptidesaccording to the present invention for use in the treatment ofproliferative diseases, such as, for example, acute myeloid leukemia,breast cancer, bile duct cancer, brain cancer, chronic lymphocyticleukemia, colorectal carcinoma, esophageal cancer, gallbladder cancer,gastric cancer, head and neck squamous cell carcinoma, hepatocellularcancer, melanoma, non-Hodgkin lymphoma, ovarian cancer, pancreaticcancer, prostate cancer, renal cell cancer, urinary bladder cancer,uterine cancer.

Particularly preferred are the peptides—alone or incombination—according to the present invention selected from the groupconsisting of SEQ ID NO: 1 to SEQ ID NO: 489. More preferred are thepeptides—alone or in combination—selected from the group consisting ofSEQ ID NO: 1 to SEQ ID NO: 374 (see Table 1), and their uses in theimmunotherapy of lung cancer (including NSCLC and SCLC), acute myeloidleukemia, breast cancer, bile duct cancer, brain cancer, chroniclymphocytic leukemia, colorectal carcinoma, esophageal cancer,gallbladder cancer, gastric cancer, head and neck squamous cellcarcinoma, hepatocellular cancer, melanoma, non-Hodgkin lymphoma,ovarian cancer, pancreatic cancer, prostate cancer, renal cell cancer,urinary bladder cancer, uterine cancer, and preferably lung cancer(including NSCLC and SCLC).

Thus, another aspect of the present invention relates to the use of thepeptides according to the present invention for the—preferablycombined—treatment of a proliferative disease selected from the group oflung cancer (including NSCLC and SCLC), acute myeloid leukemia, breastcancer, bile duct cancer, brain cancer, chronic lymphocytic leukemia,colorectal carcinoma, esophageal cancer, gallbladder cancer, gastriccancer, head and neck squamous cell carcinoma, hepatocellular cancer,melanoma, non-Hodgkin lymphoma, ovarian cancer, pancreatic cancer,prostate cancer, renal cell cancer, urinary bladder cancer, uterinecancer.

The present invention furthermore relates to peptides according to thepresent invention that have the ability to bind to a molecule of thehuman major histocompatibility complex (MHC) class-I or—in an elongatedform, such as a length-variant—MHC class-II.

The present invention further relates to the peptides according to thepresent invention wherein said peptides (each) consist or consistessentially of an amino acid sequence according to SEQ ID NO: 1 to SEQID NO: 489.

The present invention further relates to the peptides according to thepresent invention, wherein said peptide is modified and/or includesnon-peptide bonds.

The present invention further relates to the peptides according to thepresent invention, wherein said peptide is part of a fusion protein, inparticular fused to the N-terminal amino acids of the HLA-DRantigen-associated invariant chain (li), or fused to (or into thesequence of) an antibody, such as, for example, an antibody that isspecific for dendritic cells.

The present invention further relates to a nucleic acid, encoding thepeptides according to the present invention. The present inventionfurther relates to the nucleic acid according to the present inventionthat is DNA, cDNA, PNA, RNA or combinations thereof.

The present invention further relates to an expression vector capable ofexpressing and/or expressing a nucleic acid according to the presentinvention.

The present invention further relates to a peptide according to thepresent invention, a nucleic acid according to the present invention oran expression vector according to the present invention for use in thetreatment of diseases and in medicine, in particular in the treatment ofcancer.

The present invention further relates to antibodies that are specificagainst the peptides according to the present invention or complexes ofsaid peptides according to the present invention with MHC, and methodsof making these.

The present invention further relates to T-cell receptors (TCRs), inparticular soluble TCR (sTCRs) and cloned TCRs engineered intoautologous or allogeneic T cells, and methods of making these, as wellas NK cells or other cells bearing said TCR or cross-reacting with saidTCRs.

The antibodies and TCRs are additional embodiments of theimmunotherapeutic use of the peptides according to the invention athand.

The present invention further relates to a host cell comprising anucleic acid according to the present invention or an expression vectoras described before. The present invention further relates to the hostcell according to the present invention that is an antigen presentingcell, and preferably is a dendritic cell.

The present invention further relates to a method for producing apeptide according to the present invention, said method comprisingculturing the host cell according to the present invention, andisolating the peptide from said host cell or its culture medium.

The present invention further relates to said method according to thepresent invention, wherein the antigen is loaded onto class I or II MHCmolecules expressed on the surface of a suitable antigen-presenting cellor artificial antigen-presenting cell by contacting a sufficient amountof the antigen with an antigen-presenting cell.

The present invention further relates to the method according to thepresent invention, wherein the antigen-presenting cell comprises anexpression vector capable of expressing or expressing said peptidecontaining SEQ ID No. 1 to SEQ ID No.: 489, preferably containing SEQ IDNo. 1 to SEQ ID No. 374, or a variant amino acid sequence.

The present invention further relates to activated T cells, produced bythe method according to the present invention, wherein said T cellselectively recognizes a cell which expresses a polypeptide comprisingan amino acid sequence according to the present invention.

The present invention further relates to a method of killing targetcells in a patient which target cells aberrantly express a polypeptidecomprising any amino acid sequence according to the present invention,the method comprising administering to the patient an effective numberof T cells as produced according to the present invention.

The present invention further relates to the use of any peptide asdescribed, the nucleic acid according to the present invention, theexpression vector according to the present invention, the cell accordingto the present invention, the activated T lymphocyte, the T cellreceptor or the antibody or other peptide- and/or peptide-MHC-bindingmolecules according to the present invention as a medicament or in themanufacture of a medicament. Preferably, said medicament is activeagainst cancer.

Preferably, said medicament is a cellular therapy, a vaccine or aprotein based on a soluble TCR or antibody.

The present invention further relates to a use according to the presentinvention, wherein said cancer cells are lung cancer (including NSCLCand SCLC), acute myeloid leukemia, breast cancer, bile duct cancer,brain cancer, chronic lymphocytic leukemia, colorectal carcinoma,esophageal cancer, gallbladder cancer, gastric cancer, head and necksquamous cell carcinoma, hepatocellular cancer, melanoma, non-Hodgkinlymphoma, ovarian cancer, pancreatic cancer, prostate cancer, renal cellcancer, urinary bladder cancer, uterine cancer, and preferably lungcancer (including NSCLC and SCLC) cells.

The present invention further relates to biomarkers based on thepeptides according to the present invention, herein called “targets”that can be used in the diagnosis of cancer, preferably lung cancer(including NSCLC and SCLC). The marker can be over-presentation of thepeptide(s) themselves, or over-expression of the corresponding gene(s).The markers may also be used to predict the probability of success of atreatment, preferably an immunotherapy, and most preferred animmunotherapy targeting the same target that is identified by thebiomarker. For example, an antibody or soluble TCR can be used to stainsections of the tumor to detect the presence of a peptide of interest incomplex with MHC.

Optionally the antibody carries a further effector function such as animmune stimulating domain or toxin.

The present invention also relates to the use of these novel targets inthe context of cancer treatment.

DETAILED DESCRIPTION OF A PREFERRED EMBODIMENT

Stimulation of an immune response is dependent upon the presence ofantigens recognized as foreign by the host immune system. The discoveryof the existence of tumor associated antigens has raised the possibilityof using a host's immune system to intervene in tumor growth. Variousmechanisms of harnessing both the humoral and cellular arms of theimmune system are currently being explored for cancer immunotherapy.

Specific elements of the cellular immune response are capable ofspecifically recognizing and destroying tumor cells. The isolation ofT-cells from tumor-infiltrating cell populations or from peripheralblood suggests that such cells play an important role in natural immunedefense against cancer. CD8-positive T-cells, which recognize class Imolecules of the major histocompatibility complex (MHC)-bearing peptidesof usually 8 to 10 amino acid residues derived from proteins or defectribosomal products (DRIPS) located in the cytosol, play an importantrole in this response. The MHC-molecules of the human are alsodesignated as human leukocyte-antigens (HLA).

As used herein and except as noted otherwise all terms are defined asgiven below.

The term “T-cell response” means the specific proliferation andactivation of effector functions induced by a peptide in vitro or invivo. For MHC class I restricted cytotoxic T cells, effector functionsmay be lysis of peptide-pulsed, peptide-precursor pulsed or naturallypeptide-presenting target cells, secretion of cytokines, preferablyInterferon-gamma, TNF-alpha, or IL-2 induced by peptide, secretion ofeffector molecules, preferably granzymes or perforins induced bypeptide, or degranulation.

The term “peptide” is used herein to designate a series of amino acidresidues, connected one to the other typically by peptide bonds betweenthe alpha-amino and carbonyl groups of the adjacent amino acids. Thepeptides are preferably 9 amino acids in length, but can be as short as8 amino acids in length, and as long as 10, 11, or 12 or longer, and incase of MHC class II peptides (elongated variants of the peptides of theinvention) they can be as long as 13, 14, 15, 16, 17, 18, 19 or 20 ormore amino acids in length.

Furthermore, the term “peptide” shall include salts of a series of aminoacid residues, connected one to the other typically by peptide bondsbetween the alpha-amino and carbonyl groups of the adjacent amino acids.Preferably, the salts are pharmaceutical acceptable salts of thepeptides, such as, for example, the chloride or acetate(trifluoroacetate) salts. It has to be noted that the salts of thepeptides according to the present invention differ substantially fromthe peptides in their state(s) in vivo, as the peptides are not salts invivo.

The term “peptide” shall also include “oligopeptide”. The term“oligopeptide” is used herein to designate a series of amino acidresidues, connected one to the other typically by peptide bonds betweenthe alpha-amino and carbonyl groups of the adjacent amino acids. Thelength of the oligopeptide is not critical to the invention, as long asthe correct epitope or epitopes are maintained therein. Theoligopeptides are typically less than about 30 amino acid residues inlength, and greater than about 15 amino acids in length.

The term “polypeptide” designates a series of amino acid residues,connected one to the other typically by peptide bonds between thealpha-amino and carbonyl groups of the adjacent amino acids. The lengthof the polypeptide is not critical to the invention as long as thecorrect epitopes are maintained. In contrast to the terms peptide oroligopeptide, the term polypeptide is meant to refer to moleculescontaining more than about 30 amino acid residues.

A peptide, oligopeptide, protein or polynucleotide coding for such amolecule is “immunogenic” (and thus is an “immunogen” within the presentinvention), if it is capable of inducing an immune response. In the caseof the present invention, immunogenicity is more specifically defined asthe ability to induce a T-cell response. Thus, an “immunogen” would be amolecule that is capable of inducing an immune response, and in the caseof the present invention, a molecule capable of inducing a T-cellresponse. In another aspect, the immunogen can be the peptide, thecomplex of the peptide with MHC, oligopeptide, and/or protein that isused to raise specific antibodies or TCRs against it.

A class I T cell “epitope” requires a short peptide that is bound to aclass I MHC receptor, forming a ternary complex (MHC class I alphachain, beta-2-microglobulin, and peptide) that can be recognized by a Tcell bearing a matching T-cell receptor binding to the MHC/peptidecomplex with appropriate affinity. Peptides binding to MHC class Imolecules are typically 8-14 amino acids in length, and most typically 9amino acids in length.

In humans, there are three different genetic loci that encode MHC classI molecules (the MHC-molecules of the human are also designated humanleukocyte antigens (HLA)): HLA-A, HLA-B, and HLA-C. HLA-A*01, HLA-A*02,and HLA-B*07 are examples of different MHC class I alleles that can beexpressed from these loci.

TABLE 4 Expression frequencies F of HLA-A*02, HLA-A*01, HLA-A*03,HLA-A*24, HLA-B*07, HLA-B*08 and HLA-B*44 serotypes. Haplotypefrequencies Gf are derived from a study which used HLA-typing data froma registry of more than 6.5 million volunteer donors in the U.S.(Gragert et al., 2013). The haplotype frequency is the frequency of adistinct allele on an individual chromosome. Due to the diploid set ofchromosomes within mammalian cells, the frequency of genotypicoccurrence of this allele is higher and can be calculated employing theHardy-Weinberg principle (F = 1 − (1 − Gf)²). Calculated phenotype fromAllele Population allele frequency (F) A*02 African (N = 28557) 32.3%European Caucasian 49.3% (N = 1242890) Japanese (N = 24582) 42.7%Hispanic, S + Cent Amer. 46.1% (N = 146714) Southeast Asian (N = 27978)30.4% A*01 African (N = 28557) 10.2% European Caucasian 30.2% (N =1242890) Japanese (N = 24582) 1.8% Hispanic, S + Cent Amer. 14.0% (N =146714) Southeast Asian (N = 27978) 21.0% A*03 African (N = 28557) 14.8%European Caucasian 26.4% (N = 1242890) Japanese (N = 24582) 1.8%Hispanic, S + Cent Amer. 14.4% (N = 146714) Southeast Asian (N = 27978)10.6% A*24 African (N = 28557) 2.0% European Caucasian 8.6% (N =1242890) Japanese (N = 24582) 35.5% Hispanic, S + Cent Amer. 13.6% (N =146714) Southeast Asian (N = 27978) 16.9% B*07 African (N = 28557) 14.7%European Caucasian 25.0% (N = 1242890) Japanese (N = 24582) 11.4%Hispanic, S + Cent Amer. 12.2% (N = 146714) Southeast Asian (N = 27978)10.4% B*08 African (N = 28557) 6.0% European Caucasian 21.6% (N =1242890) Japanese (N = 24582) 1.0% Hispanic, S + Cent Amer. 7.6% (N =146714) Southeast Asian (N = 27978) 6.2% B*44 African (N = 28557) 10.6%European Caucasian 26.9% (N = 1242890) Japanese (N = 24582) 13.0%Hispanic, S + Cent Amer. 18.2% (N = 146714) Southeast Asian (N = 27978)13.1%

The peptides of the invention, preferably when included into a vaccineof the invention as described herein bind to A*02, A*01, A*03, A*24,B*07, B*08 or B*44. A vaccine may also include pan-binding MHC class IIpeptides. Therefore, the vaccine of the invention can be used to treatcancer in patients that are A*02-, A*01-, A*03-, A*24-, B*07-, B*08- orB*44-positive, whereas no selection for MHC class II allotypes isnecessary due to the pan-binding nature of these peptides.

If A*02 peptides of the invention are combined with peptides binding toanother allele, for example A*24, a higher percentage of any patientpopulation can be treated compared with addressing either MHC class Iallele alone. While in most populations less than 50% of patients couldbe addressed by either allele alone, a vaccine comprising HLA-A*24 andHLA-A*02 epitopes can treat at least 60% of patients in any relevantpopulation. Specifically, the following percentages of patients will bepositive for at least one of these alleles in various regions: USA 61%,Western Europe 62%, China 75%, South Korea 77%, Japan 86% (calculatedfrom www.allelefrequencies.net).

TABLE 5 HLA alleles coverage in European Caucasian population(calculated from (Gragert et al., 2013)). coverage (at least combinedone A- combined combined with B*07 allele) with B*07 with B*44 and B*44A*02/A*01 70% 78% 78% 84% A*02/A*03 68% 76% 76% 83% A*02/A*24 61% 71%71% 80% A*01/A*03 52% 64% 65% 75% A*01/A*24 44% 58% 59% 71% A*03/A*2440% 55% 56% 69% A*02/A*01/A*03 84% 88% 88% 91% A*02/A*01/A*24 79% 84%84% 89% A*02/A*03/A*24 77% 82% 83% 88% A*01/A*03/A*24 63% 72% 73% 81%A*02/A*01/A*03/ 90% 92% 93% 95% A*24

In a preferred embodiment, the term “nucleotide sequence” refers to aheteropolymer of deoxyribonucleotides.

The nucleotide sequence coding for a particular peptide, oligopeptide,or polypeptide may be naturally occurring or they may be syntheticallyconstructed. Generally, DNA segments encoding the peptides,polypeptides, and proteins of this invention are assembled from cDNAfragments and short oligonucleotide linkers, or from a series ofoligonucleotides, to provide a synthetic gene that is capable of beingexpressed in a recombinant transcriptional unit comprising regulatoryelements derived from a microbial or viral operon.

As used herein the term “a nucleotide coding for (or encoding) apeptide” refers to a nucleotide sequence coding for the peptideincluding artificial (man-made) start and stop codons compatible for thebiological system the sequence is to be expressed by, for example, adendritic cell or another cell system useful for the production of TCRs.

As used herein, reference to a nucleic acid sequence includes bothsingle stranded and double stranded nucleic acid. Thus, for example forDNA, the specific sequence, unless the context indicates otherwise,refers to the single strand DNA of such sequence, the duplex of suchsequence with its complement (double stranded DNA) and the complement ofsuch sequence.

The term “coding region” refers to that portion of a gene which eithernaturally or normally codes for the expression product of that gene inits natural genomic environment, i.e., the region coding in vivo for thenative expression product of the gene.

The coding region can be derived from a non-mutated (“normal”), mutatedor altered gene, or can even be derived from a DNA sequence, or gene,wholly synthesized in the laboratory using methods well known to thoseof skill in the art of DNA synthesis.

The term “expression product” means the polypeptide or protein that isthe natural translation product of the gene and any nucleic acidsequence coding equivalents resulting from genetic code degeneracy andthus coding for the same amino acid(s).

The term “fragment”, when referring to a coding sequence, means aportion of DNA comprising less than the complete coding region, whoseexpression product retains essentially the same biological function oractivity as the expression product of the complete coding region.

The term “DNA segment” refers to a DNA polymer, in the form of aseparate fragment or as a component of a larger DNA construct, which hasbeen derived from DNA isolated at least once in substantially pure form,i.e., free of contaminating endogenous materials and in a quantity orconcentration enabling identification, manipulation, and recovery of thesegment and its component nucleotide sequences by standard biochemicalmethods, for example, by using a cloning vector. Such segments areprovided in the form of an open reading frame uninterrupted by internalnon-translated sequences, or introns, which are typically present ineukaryotic genes. Sequences of non-translated DNA may be presentdownstream from the open reading frame, where the same do not interferewith manipulation or expression of the coding regions.

The term “primer” means a short nucleic acid sequence that can be pairedwith one strand of DNA and provides a free 3′-OH end at which a DNApolymerase starts synthesis of a deoxyribonucleotide chain.

The term “promoter” means a region of DNA involved in binding of RNApolymerase to initiate transcription.

The term “isolated” means that the material is removed from its originalenvironment (e.g., the natural environment, if it is naturallyoccurring). For example, a naturally-occurring polynucleotide orpolypeptide present in a living animal is not isolated, but the samepolynucleotide or polypeptide, separated from some or all of thecoexisting materials in the natural system, is isolated. Suchpolynucleotides could be part of a vector and/or such polynucleotides orpolypeptides could be part of a composition, and still be isolated inthat such vector or composition is not part of its natural environment.

The polynucleotides, and recombinant or immunogenic polypeptides,disclosed in accordance with the present invention may also be in“purified” form. The term “purified” does not require absolute purity;rather, it is intended as a relative definition, and can includepreparations that are highly purified or preparations that are onlypartially purified, as those terms are understood by those of skill inthe relevant art. For example, individual clones isolated from a cDNAlibrary have been conventionally purified to electrophoretichomogeneity. Purification of starting material or natural material to atleast one order of magnitude, preferably two or three orders, and morepreferably four or five orders of magnitude is expressly contemplated.Furthermore, a claimed polypeptide which has a purity of preferably99.999%, or at least 99.99% or 99.9%; and even desirably 99% by weightor greater is expressly encompassed.

The nucleic acids and polypeptide expression products disclosedaccording to the present invention, as well as expression vectorscontaining such nucleic acids and/or such polypeptides, may be in“enriched form”. As used herein, the term “enriched” means that theconcentration of the material is at least about 2, 5, 10, 100, or 1000times its natural concentration (for example), advantageously 0.01%, byweight, preferably at least about 0.1% by weight. Enriched preparationsof about 0.5%, 1%, 5%, 10%, and 20% by weight are also contemplated. Thesequences, constructs, vectors, clones, and other materials comprisingthe present invention can advantageously be in enriched or isolatedform. The term “active fragment” means a fragment, usually of a peptide,polypeptide or nucleic acid sequence, that generates an immune response(i.e., has immunogenic activity) when administered, alone or optionallywith a suitable adjuvant or in a vector, to an animal, such as a mammal,for example, a rabbit or a mouse, and also including a human, suchimmune response taking the form of stimulating a T-cell response withinthe recipient animal, such as a human. Alternatively, the “activefragment” may also be used to induce a T-cell response in vitro.

As used herein, the terms “portion”, “segment” and “fragment”, when usedin relation to polypeptides, refer to a continuous sequence of residues,such as amino acid residues, which sequence forms a subset of a largersequence. For example, if a polypeptide were subjected to treatment withany of the common endopeptidases, such as trypsin or chymotrypsin, theoligopeptides resulting from such treatment would represent portions,segments or fragments of the starting polypeptide. When used in relationto polynucleotides, these terms refer to the products produced bytreatment of said polynucleotides with any of the endonucleases.

In accordance with the present invention, the term “percent identity” or“percent identical”, when referring to a sequence, means that a sequenceis compared to a claimed or described sequence after alignment of thesequence to be compared (the “Compared Sequence”) with the described orclaimed sequence (the “Reference Sequence”). The percent identity isthen determined according to the following formula:

percent identity=100[1−(C/R)]

wherein C is the number of differences between the Reference Sequenceand the Compared Sequence over the length of alignment between theReference Sequence and the Compared Sequence, wherein

(i) each base or amino acid in the Reference Sequence that does not havea corresponding aligned base or amino acid in the Compared Sequence and

(ii) each gap in the Reference Sequence and

(iii) each aligned base or amino acid in the Reference Sequence that isdifferent from an aligned base or amino acid in the Compared Sequence,constitutes a difference and

(iiii) the alignment has to start at position 1 of the alignedsequences;

and R is the number of bases or amino acids in the Reference Sequenceover the length of the alignment with the Compared Sequence with any gapcreated in the Reference Sequence also being counted as a base or aminoacid.

If an alignment exists between the Compared Sequence and the ReferenceSequence for which the percent identity as calculated above is aboutequal to or greater than a specified minimum Percent Identity then theCompared Sequence has the specified minimum percent identity to theReference Sequence even though alignments may exist in which the hereinabove calculated percent identity is less than the specified percentidentity.

As mentioned above, the present invention thus provides a peptidecomprising a sequence that is selected from the group of consisting ofSEQ ID NO: 1 to SEQ ID NO: 489 or a variant thereof which is 88%homologous to SEQ ID NO: 1 to SEQ ID NO: 489, or a variant thereof thatwill induce T cells cross-reacting with said peptide. The peptides ofthe invention have the ability to bind to a molecule of the human majorhistocompatibility complex (MHC) class-I or elongated versions of saidpeptides to class II.

In the present invention, the term “homologous” refers to the degree ofidentity (see percent identity above) between sequences of two aminoacid sequences, i.e. peptide or polypeptide sequences. Theaforementioned “homology” is determined by comparing two sequencesaligned under optimal conditions over the sequences to be compared. Sucha sequence homology can be calculated by creating an alignment using,for example, the ClustalW algorithm. Commonly available sequenceanalysis software, more specifically, Vector NTI, GENETYX or other toolsare provided by public databases.

A person skilled in the art will be able to assess, whether T cellsinduced by a variant of a specific peptide will be able to cross-reactwith the peptide itself (Appay et al., 2006; Colombetti et al., 2006;Fong et al., 2001; Zaremba et al., 1997).

By a “variant” of the given amino acid sequence the inventors mean thatthe side chains of, for example, one or two of the amino acid residuesare altered (for example by replacing them with the side chain ofanother naturally occurring amino acid residue or some other side chain)such that the peptide is still able to bind to an HLA molecule insubstantially the same way as a peptide consisting of the given aminoacid sequence in consisting of SEQ ID NO: 1 to SEQ ID NO: 489. Forexample, a peptide may be modified so that it at least maintains, if notimproves, the ability to interact with and bind to the binding groove ofa suitable MHC molecule, such as HLA-A*02 or -DR, and in that way, it atleast maintains, if not improves, the ability to bind to the TCR ofactivated T cells.

These T cells can subsequently cross-react with cells and kill cellsthat express a polypeptide that contains the natural amino acid sequenceof the cognate peptide as defined in the aspects of the invention. Ascan be derived from the scientific literature and databases (Rammenseeet al., 1999; Godkin et al., 1997), certain positions of HLA bindingpeptides are typically anchor residues forming a core sequence fittingto the binding motif of the HLA receptor, which is defined by polar,electrophysical, hydrophobic and spatial properties of the polypeptidechains constituting the binding groove. Thus, one skilled in the artwould be able to modify the amino acid sequences set forth in SEQ ID NO:1 to SEQ ID NO 489, by maintaining the known anchor residues, and wouldbe able to determine whether such variants maintain the ability to bindMHC class I or II molecules. The variants of the present inventionretain the ability to bind to the TCR of activated T cells, which cansubsequently cross-react with and kill cells that express a polypeptidecontaining the natural amino acid sequence of the cognate peptide asdefined in the aspects of the invention.

The original (unmodified) peptides as disclosed herein can be modifiedby the substitution of one or more residues at different, possiblyselective, sites within the peptide chain, if not otherwise stated.Preferably those substitutions are located at the end of the amino acidchain. Such substitutions may be of a conservative nature, for example,where one amino acid is replaced by an amino acid of similar structureand characteristics, such as where a hydrophobic amino acid is replacedby another hydrophobic amino acid. Even more conservative would bereplacement of amino acids of the same or similar size and chemicalnature, such as where leucine is replaced by isoleucine. In studies ofsequence variations in families of naturally occurring homologousproteins, certain amino acid substitutions are more often tolerated thanothers, and these are often show correlation with similarities in size,charge, polarity, and hydrophobicity between the original amino acid andits replacement, and such is the basis for defining “conservativesubstitutions.”

Conservative substitutions are herein defined as exchanges within one ofthe following five groups: Group 1-small aliphatic, nonpolar or slightlypolar residues (Ala, Ser, Thr, Pro, Gly); Group 2-polar, negativelycharged residues and their amides (Asp, Asn, Glu, Gin); Group 3-polar,positively charged residues (His, Arg, Lys); Group 4-large, aliphatic,nonpolar residues (Met, Leu, lie, Val, Cys); and Group 5-large, aromaticresidues (Phe, Tyr, Trp).

Less conservative substitutions might involve the replacement of oneamino acid by another that has similar characteristics but is somewhatdifferent in size, such as replacement of an alanine by an isoleucineresidue. Highly non-conservative replacements might involve substitutingan acidic amino acid for one that is polar, or even for one that isbasic in character. Such “radical” substitutions cannot, however, bedismissed as potentially ineffective since chemical effects are nottotally predictable and radical substitutions might well give rise toserendipitous effects not otherwise predictable from simple chemicalprinciples.

Of course, such substitutions may involve structures other than thecommon L-amino acids. Thus, D-amino acids might be substituted for theL-amino acids commonly found in the antigenic peptides of the inventionand yet still be encompassed by the disclosure herein. In addition,non-standard amino acids (i.e., other than the common naturallyoccurring proteinogenic amino acids) may also be used for substitutionpurposes to produce immunogens and immunogenic polypeptides according tothe present invention.

If substitutions at more than one position are found to result in apeptide with substantially equivalent or greater antigenic activity asdefined below, then combinations of those substitutions will be testedto determine if the combined substitutions result in additive orsynergistic effects on the antigenicity of the peptide. At most, no morethan 4 positions within the peptide would be simultaneously substituted.

A peptide consisting essentially of the amino acid sequence as indicatedherein can have one or two non-anchor amino acids (see below regardingthe anchor motif) exchanged without that the ability to bind to amolecule of the human major histocompatibility complex (MHC) class-I or-II is substantially changed or is negatively affected, when compared tothe non-modified peptide. In another embodiment, in a peptide consistingessentially of the amino acid sequence as indicated herein, one or twoamino acids can be exchanged with their conservative exchange partners(see herein below) without that the ability to bind to a molecule of thehuman major histocompatibility complex (MHC) class-I or -II issubstantially changed, or is negatively affected, when compared to thenon-modified peptide.

The amino acid residues that do not substantially contribute tointeractions with the T-cell receptor can be modified by replacementwith other amino acid whose incorporation does not substantially affectT-cell reactivity and does not eliminate binding to the relevant MHC.Thus, apart from the proviso given, the peptide of the invention may beany peptide (by which term the inventors include oligopeptide orpolypeptide), which includes the amino acid sequences or a portion orvariant thereof as given.

TABLE 6 Variants and motif of the peptides according toSEQ ID NO: 4, 13, 90. 93, 138, 171, 202, 204,224, 294, 306, 316, 322 and 327. Position 1 2 3 4 5 6 7 8 9 SEQ ID No 4S Y E K V I N Y L Variant I F F I F F F Position 1 2 3 4 5 6 7 8 9SEQ ID No 13 H Y T Y I L E V F Variant I L F I F L F Position 1 2 3 4 56 7 8 SEQ ID No 90 F L L M H P S I Variant V L A M V M M L M A A V A A LA A V V V V L V A T V T T L T A Q V Q Q L Q A Position 1 2 3 4 5 6 7 8 9SEQ ID No 93 T L Q E K I L Q V Variant I L A M M I M L M A A A I A L A AV V I V L V A T T I T L T A Q Q I Q L Q A Position 1 2 3 4 5 6 7 8 9SEQ ID No 138 D S D S C H F N Y Variant A E E A T T A T E T E A Position1 2 3 4 5 6 7 8 9 10 11 SEQ ID No 171 R S D P V T L N V L Y Variant A EE A T T A T E T E A Position 1 2 3 4 5 6 7 8 9 10 11 SEQ ID No 202 K L AE L E G A L Q K Variant Y R F I I Y I R I F M M Y M R M F V V Y V R V FT T Y T R T F Position 1 2 3 4 5 6 7 8 9 SEQ ID No 204 A V F D K F I R YVariant L K L L R L F I K I I R I F M K M M R M F K R F T K T T R T FPosition 1 2 3 4 5 6 7 8 9 SEQ ID No 224 G P Q P W H A A L Variant F V MA I Position 1 2 3 4 5 6 7 8 9 10 SEQ ID No 294 G P T P N T G A A LVariant F V M A I Position 1 2 3 4 5 6 7 8 SEQ ID No 306 I L K V K V G LVariant V I M F R R V R I R M R F H H V H I H M H F R R V R I R M R F RR R R V R R I R R M R R F R H R H V R H I R H M R H F L L V L I L M L FL R L R V L R I L R M L R F L H L H V L H I L H M L H F Position 1 2 3 45 6 7 8 9 SEQ ID No 316 V I K E K A L T L Variant V I M F R R V R I R MR F H H V H I H M H F R R V R I R M R F R R R R V R R I R R M R R F R HR H V R H I R H M R H F L L V L I L M L F L R L R V L R I L R M L R F LH L H V L H I L H M L H F Position 1 2 3 4 5 6 7 8 9 10 SEQ ID No 322 KE V D P A G H S Y Variant F W L D F D W D D L Position 1 2 3 4 5 6 7 8 910 SEQ ID No 327 Y E I E A R N Q V F Variant W Y L D D W D Y D L

Longer (elongated) peptides may also be suitable. It is possible thatMHC class I epitopes, although usually between 8 and 11 amino acidslong, are generated by peptide processing from longer peptides orproteins that include the actual epitope. It is preferred that theresidues that flank the actual epitope are residues that do notsubstantially affect proteolytic cleavage necessary to expose the actualepitope during processing.

The peptides of the invention can be elongated by up to four aminoacids, that is 1, 2, 3 or 4 amino acids can be added to either end inany combination between 4:0 and 0:4. Combinations of the elongationsaccording to the invention can be found in Table 7.

TABLE 7 Combinations of the elongations of peptides of the inventionC-terminus N-terminus 4 0 3 0 or 1 2 0 or 1 or 2 1 0 or 1 or 2 or 3 0 0or 1 or 2 or 3 or 4 N-terminus C-terminus 4 0 3 0 or 1 2 0 or 1 or 2 1 0or 1 or 2 or 3 0 0 or 1 or 2 or 3 or 4

The amino acids for the elongation/extension can be the peptides of theoriginal sequence of the protein or any other amino acid(s). Theelongation can be used to enhance the stability or solubility of thepeptides.

Thus, the epitopes of the present invention may be identical tonaturally occurring tumor-associated or tumor-specific epitopes or mayinclude epitopes that differ by no more than four residues from thereference peptide, as long as they have substantially identicalantigenic activity.

In an alternative embodiment, the peptide is elongated on either or bothsides by more than 4 amino acids, preferably to a total length of up to30 amino acids. This may lead to MHC class II binding peptides. Bindingto MHC class II can be tested by methods known in the art.

Accordingly, the present invention provides peptides and variants of MHCclass I epitopes, wherein the peptide or variant has an overall lengthof from 8 and 100, from 9 and 100, from 10 and 100, from 11 and 100,from 12 and 100, preferably from 8 and 30, and from 9 and 30, from 10and 30, from 11 and 30, from 12 and 30, most preferred from 8 and 14,from 9 and 14, from 10 and 14, from 11 and 14, from 12 and 14. Thepresent invention further provides peptides and variants of MHC class Iepitopes, wherein the peptide or variant has an overall length of namely8, 9, 10, 11, 12, 13, or 14 amino acids, in case of the elongated classII binding peptides the length can also be 15, 16, 17, 18, 19, 20, 21 or22 amino acids.

Of course, the peptide or variant according to the present inventionwill have the ability to bind to a molecule of the human majorhistocompatibility complex (MHC) class I or II. Binding of a peptide ora variant to a MHC complex may be tested by methods known in the art.

Preferably, when the T cells specific for a peptide according to thepresent invention are tested against the substituted peptides, thepeptide concentration at which the substituted peptides achieve half themaximal increase in lysis relative to background is no more than about 1mM, preferably no more than about 1 μM, more preferably no more thanabout 1 nM, and still more preferably no more than about 100 μM, andmost preferably no more than about 10 μM. It is also preferred that thesubstituted peptide be recognized by T cells from more than oneindividual, at least two, and more preferably three individuals.

In a particularly preferred embodiment of the invention the peptideconsists or consists essentially of an amino acid sequence according toSEQ ID NO: 1 to SEQ ID NO: 489.

“Consisting essentially of” shall mean that a peptide according to thepresent invention, in addition to the sequence according to any of SEQID NO: 1 to SEQ ID NO 489 or a variant thereof contains additional N-and/or C-terminally located stretches of amino acids that are notnecessarily forming part of the peptide that functions as an epitope forMHC molecules epitope.

Nevertheless, these stretches can be important to provide an efficientintroduction of the peptide according to the present invention into thecells. In one embodiment of the present invention, the peptide is partof a fusion protein which comprises, for example, the 80 N-terminalamino acids of the HLA-DR antigen-associated invariant chain (p33, inthe following “li”) as derived from the NCBI, GenBank Accession numberX00497. In other fusions, the peptides of the present invention can befused to an antibody as described herein, or a functional part thereof,in particular into a sequence of an antibody, so as to be specificallytargeted by said antibody, or, for example, to or into an antibody thatis specific for dendritic cells as described herein.

In addition, the peptide or variant may be modified further to improvestability and/or binding to MHC molecules in order to elicit a strongerimmune response. Methods for such an optimization of a peptide sequenceare well known in the art and include, for example, the introduction ofreverse peptide bonds or non-peptide bonds.

In a reverse peptide bond, amino acid residues are not joined by peptide(—CO—NH—) linkages but the peptide bond is reversed. Such retro-inversopeptidomimetics may be made using methods known in the art, for examplesuch as those described in Meziere et al (1997) (Meziere et al., 1997),incorporated herein by reference. This approach involves makingpseudopeptides containing changes involving the backbone, and not theorientation of side chains. Meziere et al. (Meziere et al., 1997) showthat for MHC binding and T helper cell responses, these pseudopeptidesare useful. Retro-inverse peptides, which contain NH—CO bonds instead ofCO—NH peptide bonds, are much more resistant to proteolysis.

A non-peptide bond is, for example, —CH₂—NH, —CH₂S—, —CH₂CH₂—, —CH═CH—,—COCH₂—, —CH(OH)CH₂—, and —CH₂SO—. U.S. Pat. No. 4,897,445 provides amethod for the solid phase synthesis of non-peptide bonds (—CH₂—NH) inpolypeptide chains which involves polypeptides synthesized by standardprocedures and the non-peptide bond synthesized by reacting an aminoaldehyde and an amino acid in the presence of NaCNBH₃.

Peptides comprising the sequences described above may be synthesizedwith additional chemical groups present at their amino and/or carboxytermini, to enhance the stability, bioavailability, and/or affinity ofthe peptides. For example, hydrophobic groups such as carbobenzoxyl,dansyl, or t-butyloxycarbonyl groups may be added to the peptides' aminotermini. Likewise, an acetyl group or a 9-fluorenylmethoxy-carbonylgroup may be placed at the peptides' amino termini. Additionally, thehydrophobic group, t-butyloxycarbonyl, or an amido group may be added tothe peptides' carboxy termini.

Further, the peptides of the invention may be synthesized to alter theirsteric configuration. For example, the D-isomer of one or more of theamino acid residues of the peptide may be used, rather than the usualL-isomer. Still further, at least one of the amino acid residues of thepeptides of the invention may be substituted by one of the well-knownnon-naturally occurring amino acid residues. Alterations such as thesemay serve to increase the stability, bioavailability and/or bindingaction of the peptides of the invention.

Similarly, a peptide or variant of the invention may be modifiedchemically by reacting specific amino acids either before or aftersynthesis of the peptide. Examples for such modifications are well knownin the art and are summarized e.g. in R. Lundblad, Chemical Reagents forProtein Modification, 3rd ed. CRC Press, 2004 (Lundblad, 2004), which isincorporated herein by reference. Chemical modification of amino acidsincludes but is not limited to, modification by acylation, amidination,pyridoxylation of lysine, reductive alkylation, trinitrobenzylation ofamino groups with 2,4,6-trinitrobenzene sulphonic acid (TNBS), amidemodification of carboxyl groups and sulphydryl modification by performicacid oxidation of cysteine to cysteic acid, formation of mercurialderivatives, formation of mixed disulphides with other thiol compounds,reaction with maleimide, carboxymethylation with iodoacetic acid oriodoacetamide and carbamoylation with cyanate at alkaline pH, althoughwithout limitation thereto. In this regard, the skilled person isreferred to Chapter 15 of Current Protocols In Protein Science, Eds.Coligan et al. (John Wiley and Sons NY 1995-2000) (Coligan et al., 1995)for more extensive methodology relating to chemical modification ofproteins.

Briefly, modification of e.g. arginyl residues in proteins is oftenbased on the reaction of vicinal dicarbonyl compounds such asphenylglyoxal, 2,3-butanedione, and 1,2-cyclohexanedione to form anadduct. Another example is the reaction of methylglyoxal with arginineresidues. Cysteine can be modified without concomitant modification ofother nucleophilic sites such as lysine and histidine. As a result, alarge number of reagents are available for the modification of cysteine.The websites of companies such as Sigma-Aldrich (ww w.sigma-aldrich.com)provide information on specific reagents.

Selective reduction of disulfide bonds in proteins is also common.Disulfide bonds can be formed and oxidized during the heat treatment ofbiopharmaceuticals. Woodward's Reagent K may be used to modify specificglutamic acid residues. N-(3-(dimethylamino)propyl)-N′-ethylcarbodiimidecan be used to form intra-molecular crosslinks between a lysine residueand a glutamic acid residue. For example, diethylpyrocarbonate is areagent for the modification of histidyl residues in proteins. Histidinecan also be modified using 4-hydroxy-2-nonenal. The reaction of lysineresidues and other α-amino groups is, for example, useful in binding ofpeptides to surfaces or the cross-linking of proteins/peptides. Lysineis the site of attachment of poly(ethylene)glycol and the major site ofmodification in the glycosylation of proteins. Methionine residues inproteins can be modified with e.g. iodoacetamide, bromoethylamine, andchloramine T.

Tetranitromethane and N-acetylimidazole can be used for the modificationof tyrosyl residues. Cross-linking via the formation of dityrosine canbe accomplished with hydrogen peroxide/copper ions.

Recent studies on the modification of tryptophan have usedN-bromosuccinimide, 2-hydroxy-5-nitrobenzyl bromide or3-bromo-3-methyl-2-(2-nitrophenylmercapto)-3H-indole (BPNS-skatole).

Successful modification of therapeutic proteins and peptides with PEG isoften associated with an extension of circulatory half-life whilecross-linking of proteins with glutaraldehyde, polyethylene glycoldiacrylate and formaldehyde is used for the preparation of hydrogels.

Chemical modification of allergens for immunotherapy is often achievedby carbamylation with potassium cyanate.

A peptide or variant, wherein the peptide is modified or includesnon-peptide bonds is a preferred embodiment of the invention.

Another embodiment of the present invention relates to a non-naturallyoccurring peptide wherein said peptide consists or consists essentiallyof an amino acid sequence according to SEQ ID NO: 1 to SEQ ID NO: 489and has been synthetically produced (e.g. synthesized) as apharmaceutically acceptable salt. Methods to synthetically producepeptides are well known in the art. The salts of the peptides accordingto the present invention differ substantially from the peptides in theirstate(s) in vivo, as the peptides as generated in vivo are no salts. Thenon-natural salt form of the peptide mediates the solubility of thepeptide, in particular in the context of pharmaceutical compositionscomprising the peptides, e.g. the peptide vaccines as disclosed herein.A sufficient and at least substantial solubility of the peptide(s) isrequired in order to efficiently provide the peptides to the subject tobe treated. Preferably, the salts are pharmaceutically acceptable saltsof the peptides. These salts according to the invention include alkalineand earth alkaline salts such as salts of the Hofmeister seriescomprising as anions PO₄ ³⁻, SO₄ ²⁻, CH₃COO⁻, Cl⁻, Br⁻, NO₃ ⁻, ClO₄ ⁻,I⁻, SCN⁻ and as cations NH₄ ⁺, Rb⁺, K⁺, Na⁺, Cs⁺, Li⁺, Zn²⁺, Mg²⁺, Ca²⁺,Mn²⁺, Cu²⁺ and Ba²⁺. Particularly salts are selected from (NH₄)₃PO₄,(NH₄)₂HPO₄, (NH₄)H₂PO₄, (NH₄)₂SO₄, NH₄CH₃COO, NH₄Cl, NH₄Br, NH₄NO₃,NH₄ClO₄, NH₄I, NH₄SCN, Rb₃PO₄, Rb₂HPO₄, RbH₂PO₄, Rb₂SO₄, Rb₄CH₃COO,Rb₄Cl, Rb₄Br, Rb₄NO₃, Rb₄ClO₄, Rb₄₁, Rb₄SCN, K₃PO₄, K₂HPO₄, KH₂PO₄,K₂SO₄, KCH₃COO, KCl, KBr, KNO₃, KClO₄, KI, KSCN, Na₃PO₄, Na₂HPO₄,NaH₂PO₄, Na₂SO₄, NaCH₃COO, NaCl, NaBr, NaNO₃, NaClO₄, NaI, NaSCN, ZnCl₂Cs₃PO₄, Cs₂HPO₄, CsH₂PO₄, Cs₂SO₄, CsCH₃COO, CsCl, CsBr, CsNO₃, CsClO₄,CsI, CsSCN, Li₃PO₄, Li₂HPO₄, LiH₂PO₄, Li₂SO₄, LiCH₃COO, LiCl, LiBr,LiNO₃, LiClO₄, LiI, LiSCN, Cu₂SO₄, Mg₃(PO₄)₂, Mg₂HPO₄, Mg(H₂PO₄)₂,Mg₂SO₄, Mg(CH₃COO)₂, MgCl₂, MgBr₂, Mg(NO₃)₂, Mg(ClO₄)₂, MgI₂, Mg(SCN)₂,MnCl₂, Ca₃(PO₄), Ca₂HPO₄, Ca(H₂PO₄)₂, CaSO₄, Ca(CH₃COO)₂, CaCl₂, CaBr₂,Ca(NO₃)₂, Ca(ClO₄)₂, CaI₂, Ca(SCN)₂, Ba₃(PO₄)₂, Ba₂HPO₄, Ba(H₂PO₄)₂,BaSO₄, Ba(CH₃COO)₂, BaCl₂, BaBr₂, Ba(NO₃)₂, Ba(ClO₄)₂, BaI₂, andBa(SCN)₂. Particularly preferred are NH acetate, MgCl₂, KH₂PO₄, Na₂SO₄,KCl, NaCl, and CaCl₂, such as, for example, the chloride or acetate(trifluoroacetate) salts.

Generally, peptides and variants (at least those containing peptidelinkages between amino acid residues) may be synthesized by theFmoc-polyamide mode of solid-phase peptide synthesis as disclosed byLukas et al. (Lukas et al., 1981) and by references as cited therein.Temporary N-amino group protection is afforded by the9-fluorenylmethyloxycarbonyl (Fmoc) group. Repetitive cleavage of thishighly base-labile protecting group is done using 20% piperidine in N,N-dimethylformamide. Side-chain functionalities may be protected astheir butyl ethers (in the case of serine threonine and tyrosine), butylesters (in the case of glutamic acid and aspartic acid),butyloxycarbonyl derivative (in the case of lysine and histidine),trityl derivative (in the case of cysteine) and4-methoxy-2,3,6-trimethylbenzenesulphonyl derivative (in the case ofarginine). Where glutamine or asparagine are C-terminal residues, use ismade of the 4,4′-dimethoxybenzhydryl group for protection of the sidechain amido functionalities. The solid-phase support is based on apolydimethyl-acrylamide polymer constituted from the three monomersdimethylacrylamide (backbone-monomer), bisacryloylethylene diamine(cross linker) and acryloylsarcosine methyl ester (functionalizingagent). The peptide-to-resin cleavable linked agent used is theacid-labile 4-hydroxymethyl-phenoxyacetic acid derivative. All aminoacid derivatives are added as their preformed symmetrical anhydridederivatives with the exception of asparagine and glutamine, which areadded using a reversed N, N-dicyclohexyl-carbodiimide/1hydroxybenzotriazole mediated coupling procedure. All coupling anddeprotection reactions are monitored using ninhydrin, trinitrobenzenesulphonic acid or isotin test procedures. Upon completion of synthesis,peptides are cleaved from the resin support with concomitant removal ofside-chain protecting groups by treatment with 95% trifluoroacetic acidcontaining a 50% scavenger mix. Scavengers commonly used includeethanedithiol, phenol, anisole and water, the exact choice depending onthe constituent amino acids of the peptide being synthesized. Also acombination of solid phase and solution phase methodologies for thesynthesis of peptides is possible (see, for example, (Bruckdorfer etal., 2004), and the references as cited therein).

Trifluoroacetic acid is removed by evaporation in vacuo, with subsequenttrituration with diethyl ether affording the crude peptide. Anyscavengers present are removed by a simple extraction procedure which onlyophilization of the aqueous phase affords the crude peptide free ofscavengers. Reagents for peptide synthesis are generally available frome.g. Calbiochem-Novabiochem (Nottingham, UK).

Purification may be performed by any one, or a combination of,techniques such as re-crystallization, size exclusion chromatography,ion-exchange chromatography, hydrophobic interaction chromatography and(usually) reverse-phase high performance liquid chromatography usinge.g. acetonitrile/water gradient separation.

Analysis of peptides may be carried out using thin layer chromatography,electrophoresis, in particular capillary electrophoresis, solid phaseextraction (CSPE), reverse-phase high performance liquid chromatography,amino-acid analysis after acid hydrolysis and by fast atom bombardment(FAB) mass spectrometric analysis, as well as MALDI and ESI-Q-TOF massspectrometric analysis.

In order to select over-presented peptides, a presentation profile iscalculated showing the median sample presentation as well as replicatevariation. The profile juxtaposes samples of the tumor entity ofinterest to a baseline of normal tissue samples. Each of these profilescan then be consolidated into an over-presentation score by calculatingthe p-value of a Linear Mixed-Effects Model (Pinheiro et al., 2015)adjusting for multiple testing by False Discovery Rate (Benjamini andHochberg, 1995) (cf. Example 1, FIGS. 1A through 1N).

For the identification and relative quantitation of HLA ligands by massspectrometry, HLA molecules from shock-frozen tissue samples werepurified and HLA-associated peptides were isolated. The isolatedpeptides were separated and sequences were identified by onlinenano-electrospray-ionization (nanoESI) liquid chromatography-massspectrometry (LC-MS) experiments. The resulting peptide sequences wereverified by comparison of the fragmentation pattern of naturaltumor-associated peptides (TUMAPs) recorded from lung cancer (includingNSCLC and SCLC) samples (N=201 samples) with the fragmentation patternsof corresponding synthetic reference peptides of identical sequences.Since the peptides were directly identified as ligands of HLA moleculesof primary tumors, these results provide direct evidence for the naturalprocessing and presentation of the identified peptides on primary cancertissue obtained from 201 lung cancer (including NSCLC and SCLC)patients.

The discovery pipeline XPRESIDENT® v2.1 (see, for example, US2013-0096016, which is hereby incorporated by reference in its entirety)allows the identification and selection of relevant over-presentedpeptide vaccine candidates based on direct relative quantitation ofHLA-restricted peptide levels on cancer tissues in comparison to severaldifferent non-cancerous tissues and organs. This was achieved by thedevelopment of label-free differential quantitation using the acquiredLC-MS data processed by a proprietary data analysis pipeline, combiningalgorithms for sequence identification, spectral clustering, ioncounting, retention time alignment, charge state deconvolution andnormalization.

Presentation levels including error estimates for each peptide andsample were established. Peptides exclusively presented on tumor tissueand peptides over-presented in tumor versus non-cancerous tissues andorgans have been identified.

HLA-peptide complexes from lung cancer (including NSCLC and SCLC) tissuesamples were purified and HLA-associated peptides were isolated andanalyzed by LC-MS (see example 1). All TUMAPs contained in the presentapplication were identified with this approach on lung cancer (includingNSCLC and SCLC) samples confirming their presentation on lung cancer(including NSCLC and SCLC).

TUMAPs identified on multiple lung cancer (including NSCLC and SCLC) andnormal tissues were quantified using ion-counting of label-free LC-MSdata. The method assumes that LC-MS signal areas of a peptide correlatewith its abundance in the sample. All quantitative signals of a peptidein various LC-MS experiments were normalized based on central tendency,averaged per sample and merged into a bar plot, called presentationprofile. The presentation profile consolidates different analysismethods like protein database search, spectral clustering, charge statedeconvolution (decharging) and retention time alignment andnormalization.

Besides over-presentation of the peptide, mRNA expression of theunderlying gene was tested. mRNA data were obtained via RNASeq analysesof normal tissues and cancer tissues (cf. Example 2, FIGS. 2A through2N). An additional source of normal tissue data was a database ofpublicly available RNA expression data from around 3000 normal tissuesamples (Lonsdale, 2013). Peptides which are derived from proteins whosecoding mRNA is highly expressed in cancer tissue, but very low or absentin vital normal tissues, were preferably included in the presentinvention.

The present invention provides peptides that are useful in treatingcancers/tumors, preferably lung cancer (including NSCLC and SCLC) thatover- or exclusively present the peptides of the invention. Thesepeptides were shown by mass spectrometry to be naturally presented byHLA molecules on primary human lung cancer (including NSCLC and SCLC)samples.

Many of the source gene/proteins (also designated “full-length proteins”or “underlying proteins”) from which the peptides are derived were shownto be highly over-expressed in cancer compared with normaltissues—“normal tissues” in relation to this invention shall mean eitherhealthy lung cells or other normal tissue cells, demonstrating a highdegree of tumor association of the source genes (see Example 2).Moreover, the peptides themselves are strongly over-presented on tumortissue—“tumor tissue” in relation to this invention shall mean a samplefrom a patient suffering from lung cancer (including NSCLC and SCLC),but not on normal tissues (see Example 1).

HLA-bound peptides can be recognized by the immune system, specificallyT lymphocytes. T cells can destroy the cells presenting the recognizedHLA/peptide complex, e.g. lung cancer (including NSCLC and SCLC) cellspresenting the derived peptides.

The peptides of the present invention have been shown to be capable ofstimulating T cell responses and/or are over-presented and thus can beused for the production of antibodies and/or TCRs, such as soluble TCRs,according to the present invention (see Example 3, Example 4).Furthermore, the peptides when complexed with the respective MHC can beused for the production of antibodies and/or TCRs, in particular sTCRs,according to the present invention, as well. Respective methods are wellknown to the person of skill, and can be found in the respectiveliterature as well (see also below).

Thus, the peptides of the present invention are useful for generating animmune response in a patient by which tumor cells can be destroyed. Animmune response in a patient can be induced by direct administration ofthe described peptides or suitable precursor substances (e.g. elongatedpeptides, proteins, or nucleic acids encoding these peptides) to thepatient, ideally in combination with an agent enhancing theimmunogenicity (i.e. an adjuvant). The immune response originating fromsuch a therapeutic vaccination can be expected to be highly specificagainst tumor cells because the target peptides of the present inventionare not presented on normal tissues in comparable copy numbers,preventing the risk of undesired autoimmune reactions against normalcells in the patient.

The present description further relates to T-cell receptors (TCRs)comprising an alpha chain and a beta chain (“alpha/beta TCRs”). Alsoprovided are peptides according to the invention capable of binding toTCRs and antibodies when presented by an MHC molecule.

The present description also relates to fragments of the TCRs accordingto the invention that are capable of binding to a peptide antigenaccording to the present invention when presented by an HLA molecule.The term particularly relates to soluble TCR fragments, for example TCRsmissing the transmembrane parts and/or constant regions, single chainTCRs, and fusions thereof to, for example, with Ig.

The present description also relates to nucleic acids, vectors and hostcells for expressing TCRs and peptides of the present description; andmethods of using the same.

The term “T-cell receptor” (abbreviated TCR) refers to a heterodimericmolecule comprising an alpha polypeptide chain (alpha chain) and a betapolypeptide chain (beta chain), wherein the heterodimeric receptor iscapable of binding to a peptide antigen presented by an HLA molecule.The term also includes so-called gamma/delta TCRs.

In one embodiment the desembodiment, provides a method of producing aTCR as described herein, the method comprising culturing a host cellcapable of expressing the TCR under conditions suitable to promoteexpression of the TCR.

The description in another aspect relates to methods according to thedescription, wherein the antigen is loaded onto class I or II MHCmolecules expressed on the surface of a suitable antigen-presenting cellor artificial antigen-presenting cell by contacting a sufficient amountof the antigen with an antigen-presenting cell or the antigen is loadedonto class I or II MHC tetramers by tetramerizing the antigen/class I orII MHC complex monomers.

The alpha and beta chains of alpha/beta TCR's, and the gamma and deltachains of gamma/delta TCRs, are generally regarded as each having two“domains”, namely variable and constant domains. The variable domainconsists of a concatenation of variable region (V), and joining region(J). The variable domain may also include a leader region (L). Beta anddelta chains may also include a diversity region (D). The alpha and betaconstant domains may also include C-terminal transmembrane (TM) domainsthat anchor the alpha and beta chains to the cell membrane.

With respect to gamma/delta TCRs, the term “TCR gamma variable domain”as used herein refers to the concatenation of the TCR gamma V (TRGV)region without leader region (L), and the TCR gamma J (TRGJ) region, andthe term TCR gamma constant domain refers to the extracellular TRGCregion, or to a C-terminal truncated TRGC sequence. Likewise, the term“TCR delta variable domain” refers to the concatenation of the TCR deltaV (TRDV) region without leader region (L) and the TCR delta D/J(TRDD/TRDJ) region, and the term “TCR delta constant domain” refers tothe extracellular TRDC region, or to a C-terminal truncated TRDCsequence.

TCRs of the present description preferably bind to a peptide-HLAmolecule complex with a binding affinity (KD) of about 100 μM or less,about 50 μM or less, about 25 μM or less, or about 10 μM or less. Morepreferred are high affinity TCRs having binding affinities of about 1 μMor less, about 100 nM or less, about 50 nM or less, about 25 nM or less.Non-limiting examples of preferred binding affinity ranges for TCRs ofthe present invention include about 1 nM to about 10 nM; about 10 nM toabout 20 nM; about 20 nM to about 30 nM; about 30 nM to about 40 nM;about 40 nM to about 50 nM; about 50 nM to about 60 nM; about 60 nM toabout 70 nM; about 70 nM to about 80 nM; about 80 nM to about 90 nM; andabout 90 nM to about 100 nM.

As used herein in connect with TCRs of the present description,“specific binding” and grammatical variants thereof are used to mean aTCR having a binding affinity (KD) for a peptide-HLA molecule complex of100 μM or less.

Alpha/beta heterodimeric TCRs of the present description may have anintroduced disulfide bond between their constant domains. Preferred TCRsof this type include those which have a TRAC constant domain sequenceand a TRBC1 or TRBC2 constant domain sequence except that Thr 48 of TRACand Ser 57 of TRBC1 or TRBC2 are replaced by cysteine residues, the saidcysteines forming a disulfide bond between the TRAC constant domainsequence and the TRBC1 or TRBC2 constant domain sequence of the TCR.

With or without the introduced inter-chain bond mentioned above,alpha/beta hetero-dimeric TCRs of the present description may have aTRAC constant domain sequence and a TRBC1 or TRBC2 constant domainsequence, and the TRAC constant domain sequence and the TRBC1 or TRBC2constant domain sequence of the TCR may be linked by the nativedisulfide bond between Cys4 of exon 2 of TRAC and Cys2 of exon 2 ofTRBC1 or TRBC2.

TCRs of the present description may comprise a detectable label selectedfrom the group consisting of a radionuclide, a fluorophore and biotin.TCRs of the present description may be conjugated to a therapeuticallyactive agent, such as a radionuclide, a chemotherapeutic agent, or atoxin.

In an embodiment, a TCR of the present description having at least onemutation in the alpha chain and/or having at least one mutation in thebeta chain has modified glycosylation compared to the unmutated TCR.

In an embodiment, a TCR comprising at least one mutation in the TCRalpha chain and/or TCR beta chain has a binding affinity for, and/or abinding half-life for, a peptide-HLA molecule complex, which is at leastdouble that of a TCR comprising the unmutated TCR alpha chain and/orunmutated TCR beta chain. Affinity-enhancement of tumor-specific TCRs,and its exploitation, relies on the existence of a window for optimalTCR affinities. The existence of such a window is based on observationsthat TCRs specific for HLA-A2-restricted pathogens have KD values thatare generally about 10-fold lower when compared to TCRs specific forHLA-A2-restricted tumor-associated self-antigens. For TCRs specific forpatogenes restricted to other alleles compared to tumor-associatedself-antigens the KD values might be in a slightly different range, butthere are no general differences between the different alleles withrespect to the possibility of creating TCRs. It is now known, althoughtumor antigens have the potential to be immunogenic, because tumorsarise from the individual's own cells only mutated proteins or proteinswith altered translational processing will be seen as foreign by theimmune system. Antigens that are upregulated or overexpressed (so calledself-antigens) will not necessarily induce a functional immune responseagainst the tumor: T-cells expressing TCRs that are highly reactive tothese antigens will have been negatively selected within the thymus in aprocess known as central tolerance, meaning that only T-cells withlow-affinity TCRs for self-antigens remain. Therefore, affinity of TCRsor variants of the present description to peptides can be enhanced bymethods well known in the art.

The present description further relates to a method of identifying andisolating a TCR according to the present description, said methodcomprising incubating PBMCs from healthy donors negative with respect tothe allel at hand with the HLA/peptide monomers, incubating the PBMCswith tetramer-phycoerythrin (PE) and isolating the high avidity T-cellsby fluorescence activated cell sorting (FACS)-Calibur analysis.

The present description further relates to a method of identifying andisolating a TCR according to the present description, said methodcomprising obtaining a transgenic mouse with the entire human TCRαβ geneloci (1.1 and 0.7 Mb), whose T-cells express a diverse human TCRrepertoire that compensates for mouse TCR deficiency, immunizing themouse with a peptide, incubating PBMCs obtained from the transgenic micewith tetramer-phycoerythrin (PE), and isolating the high avidity T-cellsby fluorescence activated cell sorting (FACS)-Calibur analysis.

In one aspect, to obtain T-cells expressing TCRs of the presentdescription, nucleic acids encoding TCR-alpha and/or TCR-beta chains ofthe present description are cloned into expression vectors, such asgamma retrovirus or lentivirus. The recombinant viruses are generatedand then tested for functionality, such as antigen specificity andfunctional avidity. An aliquot of the final product is then used totransduce the target T-cell population (generally purified from patientPBMCs), which is expanded before infusion into the patient.

In another aspect, to obtain T-cells expressing TCRs of the presentdescription, TCR RNAs are synthesized by techniques known in the art,e.g., in vitro transcription systems. The in vitro-synthesized TCR RNAsare then introduced into primary CD8+ T-cells obtained from healthydonors by electroporation to re-express tumor specific TCR-alpha and/orTCR-beta chains.

To increase the expression, nucleic acids encoding TCRs of the presentdescription may be operably linked to strong promoters, such asretroviral long terminal repeats (LTRs), cytomegalovirus (CMV), murinestem cell virus (MSCV) U3, phosphoglycerate kinase (PGK), β-actin,ubiquitin, and a simian virus 40 (SV40)/CD43 composite promoter,elongation factor (EF)-1a and the spleen focus-forming virus (SFFV)promoter. In a preferred embodiment, the promoter is heterologous to thenucleic acid being expressed.

In addition to strong promoters, TCR expression cassettes of the presentdescription may contain additional elements that can enhance transgeneexpression, including a central polypurine tract (cPPT), which promotesthe nuclear translocation of lentiviral constructs (Follenzi et al.,2000), and the woodchuck hepatitis virus posttranscriptional regulatoryelement (wPRE), which increases the level of transgene expression byincreasing RNA stability (Zufferey et al., 1999).

The alpha and beta chains of a TCR of the present invention may beencoded by nucleic acids located in separate vectors, or may be encodedby polynucleotides located in the same vector.

Achieving high-level TCR surface expression requires that both theTCR-alpha and TCR-beta chains of the introduced TCR be transcribed athigh levels. To do so, the TCR-alpha and TCR-beta chains of the presentdescription may be cloned into bi-cistronic constructs in a singlevector, which has been shown to be capable of over-coming this obstacle.The use of a viral intraribosomal entry site (IRES) between theTCR-alpha and TCR-beta chains results in the coordinated expression ofboth chains, because the TCR-alpha and TCR-beta chains are generatedfrom a single transcript that is broken into two proteins duringtranslation, ensuring that an equal molar ratio of TCR-alpha andTCR-beta chains are produced (Schmitt et al., 2009).

Nucleic acids encoding TCRs of the present description may be codonoptimized to increase expression from a host cell. Redundancy in thegenetic code allows some amino acids to be encoded by more than onecodon, but certain codons are less “op-timal” than others because of therelative availability of matching tRNAs as well as other factors(Gustafsson et al., 2004). Modifying the TCR-alpha and TCR-beta genesequences such that each amino acid is encoded by the optimal codon formammalian gene expression, as well as eliminating mRNA instabilitymotifs or cryptic splice sites, has been shown to significantly enhanceTCR-alpha and TCR-beta gene expression (Scholten et al., 2006).

Furthermore, mispairing between the introduced and endogenous TCR chainsmay result in the acquisition of specificities that pose a significantrisk for autoimmunity. For example, the formation of mixed TCR dimersmay reduce the number of CD3 molecules available to form properly pairedTCR complexes, and therefore can significantly decrease the functionalavidity of the cells expressing the introduced TCR (Kuball et al.,2007).

To reduce mispairing, the C-terminus domain of the introduced TCR chainsof the present description may be modified in order to promoteinterchain affinity, while de-creasing the ability of the introducedchains to pair with the endogenous TCR. These strategies may includereplacing the human TCR-alpha and TCR-beta C-terminus domains with theirmurine counterparts (murinized C-terminus domain); generating a secondinterchain disulfide bond in the C-terminus domain by introducing asecond cysteine residue into both the TCR-alpha and TCR-beta chains ofthe introduced TCR (cysteine modification); swapping interactingresidues in the TCR-alpha and TCR-beta chain C-terminus domains(“knob-in-hole”); and fusing the variable domains of the TCR-alpha andTCR-beta chains directly to CD3 (CD3 fusion) (Schmitt et al., 2009).

In an embodiment, a host cell is engineered to express a TCR of thepresent description. In preferred embodiments, the host cell is a humanT-cell or T-cell progenitor. In some embodiments, the T-cell or T-cellprogenitor is obtained from a cancer patient. In other embodiments, theT-cell or T-cell progenitor is obtained from a healthy donor. Host cellsof the present description can be allogeneic or autologous with respectto a patient to be treated. In one embodiment, the host is a gamma/deltaT-cell transformed to express an alpha/beta TCR.

A “pharmaceutical composition” is a composition suitable foradministration to a human being in a medical setting. Preferably, apharmaceutical composition is sterile and produced according to GMPguidelines.

The pharmaceutical compositions comprise the peptides either in the freeform or in the form of a pharmaceutically acceptable salt (see alsoabove). In an aspect, a peptide described herein is in the form of apharmaceutically acceptable salt. In another aspect, a peptide in theform of a pharmeutical salt is in crystalline form.

In an aspect, a pharmaceutically acceptable salt described herein refersto salts which possess toxicity profiles within a range that isacceptable for pharmaceutical applications.

As used herein, “a pharmaceutically acceptable salt” refers to aderivative of the disclosed peptides wherein the peptide is modified bymaking acid or base salts of the agent. For example, acid salts areprepared from the free base (typically wherein the neutral form of thedrug has a neutral —NH2 group) involving reaction with a suitable acid.Suitable acids for preparing acid salts include both organic acids,e.g., acetic acid, propionic acid, glycolic acid, pyruvic acid, oxalicacid, malic acid, malonic acid, succinic acid, maleic acid, fumaricacid, tartaric acid, citric acid, benzoic acid, cinnamic acid, mandelicacid, methane sulfonic acid, ethane sulfonic acid, p-toluenesulfonicacid, salicylic acid, and the like, as well as inorganic acids, e.g.,hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acidphosphoric acid and the like. Conversely, preparation of basic salts ofacid moieties which may be present on a peptide are prepared using apharmaceutically acceptable base such as sodium hydroxide, potassiumhydroxide, ammonium hydroxide, calcium hydroxide, trimethylamine or thelike.

In an aspect, pharmacuetically acceptable salts may increase thesolubility and/or stability of peptides of described herein. In anotheraspect, pharmacutical salts described herein may be prepared byconventional means from the corresponding carrier peptide or complex byreacting, for example, the appropriate acid or base with peptides orcomplexes as described herein. In another aspect, the pharmacueticallyacceptable salts are in crystalline form or semi-crystalline form. Inyet another aspect, pharmaceutically acceptable salts may include, forexample, those described in Handbook of Pharmaceutical Salts:Properties, Selection, and Use By P. H. Stahl and C. G. Wermuth(Wiley-VCH 2002) and L. D. Bighley, S. M. Berge, D. C. Monkhouse, in“Encyclopedia of Pharmaceutical Technology”. Eds. J. Swarbrick and J. C.Boylan, Vol. 13, Marcel Dekker, Inc., New York, Basel, Hong Kong 1995,pp. 453-499, each of these references is herein incorporated byreference in their entirety.

In an especially preferred embodiment, the pharmaceutical compositionscomprise the peptides as salts of acetic acid (acetates), trifluoroacetates or hydrochloric acid (chlorides).

Preferably, the medicament of the present invention is animmunotherapeutic such as a vaccine. It may be administered directlyinto the patient, into the affected organ or systemically i.d., i.m.,s.c., i.p. and i.v., or applied ex vivo to cells derived from thepatient or a human cell line which are subsequently administered to thepatient, or used in vitro to select a subpopulation of immune cellsderived from the patient, which are then re-administered to the patient.If the nucleic acid is administered to cells in vitro, it may be usefulfor the cells to be transfected so as to co-express immune-stimulatingcytokines, such as interleukin-2. The peptide may be substantially pure,or combined with an immune-stimulating adjuvant (see below) or used incombination with immune-stimulatory cytokines, or be administered with asuitable delivery system, for example liposomes. The peptide may also beconjugated to a suitable carrier such as keyhole limpet haemocyanin(KLH) or mannan (see WO 95/18145 and (Longenecker et al., 1993)). Thepeptide may also be tagged, may be a fusion protein, or may be a hybridmolecule. The peptides whose sequence is given in the present inventionare expected to stimulate CD4 or CD8 T cells. However, stimulation ofCD8 T cells is more efficient in the presence of help provided by CD4T-helper cells. Thus, for MHC Class I epitopes that stimulate CD8 Tcells the fusion partner or sections of a hybrid molecule suitablyprovide epitopes which stimulate CD4-positive T cells. CD4- andCD8-stimulating epitopes are well known in the art and include thoseidentified in the present invention.

In one aspect, the vaccine comprises at least one peptide having theamino acid sequence set forth SEQ ID No. 1 to SEQ ID No. 489, and atleast one additional peptide, preferably two to 50, more preferably twoto 25, even more preferably two to 20 and most preferably two, three,four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen,fourteen, fifteen, sixteen, seventeen or eighteen peptides. Thepeptide(s) may be derived from one or more specific TAAs and may bind toMHC class I molecules.

A further aspect of the invention provides a nucleic acid (for example apolynucleotide) encoding a peptide or peptide variant of the invention.The polynucleotide may be, for example, DNA, cDNA, PNA, RNA orcombinations thereof, either single- and/or double-stranded, or nativeor stabilized forms of polynucleotides, such as, for example,polynucleotides with a phosphorothioate backbone and it may or may notcontain introns so long as it codes for the peptide. Of course, onlypeptides that contain naturally occurring amino acid residues joined bynaturally occurring peptide bonds are encodable by a polynucleotide. Astill further aspect of the invention provides an expression vectorcapable of expressing a polypeptide according to the invention.

A variety of methods have been developed to link polynucleotides,especially DNA, to vectors for example via complementary cohesivetermini. For instance, complementary homopolymer tracts can be added tothe DNA segment to be inserted to the vector DNA. The vector and DNAsegment are then joined by hydrogen bonding between the complementaryhomopolymeric tails to form recombinant DNA molecules.

Synthetic linkers containing one or more restriction sites provide analternative method of joining the DNA segment to vectors. Syntheticlinkers containing a variety of restriction endonuclease sites arecommercially available from a number of sources including InternationalBiotechnologies Inc. New Haven, Conn., USA.

A desirable method of modifying the DNA encoding the polypeptide of theinvention employs the polymerase chain reaction as disclosed by Saiki RK, et al. (Saiki et al., 1988). This method may be used for introducingthe DNA into a suitable vector, for example by engineering in suitablerestriction sites, or it may be used to modify the DNA in other usefulways as is known in the art. If viral vectors are used, pox- oradenovirus vectors are preferred.

The DNA (or in the case of retroviral vectors, RNA) may then beexpressed in a suitable host to produce a polypeptide comprising thepeptide or variant of the invention. Thus, the DNA encoding the peptideor variant of the invention may be used in accordance with knowntechniques, appropriately modified in view of the teachings containedherein, to construct an expression vector, which is then used totransform an appropriate host cell for the expression and production ofthe polypeptide of the invention. Such techniques include thosedisclosed, for example, in U.S. Pat. Nos. 4,440,859, 4,530,901,4,582,800, 4,677,063, 4,678,751, 4,704,362, 4,710,463, 4,757,006,4,766,075, and 4,810,648.

The DNA (or in the case of retroviral vectors, RNA) encoding thepolypeptide constituting the compound of the invention may be joined toa wide variety of other DNA sequences for introduction into anappropriate host. The companion DNA will depend upon the nature of thehost, the manner of the introduction of the DNA into the host, andwhether episomal maintenance or integration is desired.

Generally, the DNA is inserted into an expression vector, such as aplasmid, in proper orientation and correct reading frame for expression.If necessary, the DNA may be linked to the appropriate transcriptionaland translational regulatory control nucleotide sequences recognized bythe desired host, although such controls are generally available in theexpression vector. The vector is then introduced into the host throughstandard techniques. Generally, not all of the hosts will be transformedby the vector. Therefore, it will be necessary to select for transformedhost cells. One selection technique involves incorporating into theexpression vector a DNA sequence, with any necessary control elements,that codes for a selectable trait in the transformed cell, such asantibiotic resistance.

Alternatively, the gene for such selectable trait can be on anothervector, which is used to co-transform the desired host cell.

Host cells that have been transformed by the recombinant DNA of theinvention are then cultured for a sufficient time and under appropriateconditions known to those skilled in the art in view of the teachingsdisclosed herein to permit the expression of the polypeptide, which canthen be recovered.

Many expression systems are known, including bacteria (for example E.coli and Bacillus subtilis), yeasts (for example Saccharomycescerevisiae), filamentous fungi (for example Aspergillus spec.), plantcells, animal cells and insect cells. Preferably, the system can bemammalian cells such as CHO cells available from the ATCC Cell BiologyCollection.

A typical mammalian cell vector plasmid for constitutive expressioncomprises the CMV or SV40 promoter with a suitable poly A tail and aresistance marker, such as neomycin.

One example is pSVL available from Pharmacia, Piscataway, N.J., USA. Anexample of an inducible mammalian expression vector is pMSG, alsoavailable from Pharmacia. Useful yeast plasmid vectors are pRS403-406and pRS413-416 and are generally available from Stratagene CloningSystems, La Jolla, Calif. 92037, USA. Plasmids pRS403, pRS404, pRS405and pRS406 are Yeast Integrating plasmids (YIps) and incorporate theyeast selectable markers HIS3, TRP1, LEU2 and URA3. Plasmids pRS413-416are Yeast Centromere plasmids (Ycps). CMV promoter-based vectors (forexample from Sigma-Aldrich) provide transient or stable expression,cytoplasmic expression or secretion, and N-terminal or C-terminaltagging in various combinations of FLAG, 3×FLAG, c-myc or MAT. Thesefusion proteins allow for detection, purification and analysis ofrecombinant protein. Dual-tagged fusions provide flexibility indetection.

The strong human cytomegalovirus (CMV) promoter regulatory region drivesconstitutive protein expression levels as high as 1 mg/L in COS cells.For less potent cell lines, protein levels are typically ˜0.1 mg/L. Thepresence of the SV40 replication origin will result in high levels ofDNA replication in SV40 replication permissive COS cells. CMV vectors,for example, can contain the pMB1 (derivative of pBR322) origin forreplication in bacterial cells, the b-lactamase gene for ampicillinresistance selection in bacteria, hGH polyA, and the f1 origin. Vectorscontaining the pre-pro-trypsin leader (PPT) sequence can direct thesecretion of FLAG fusion proteins into the culture medium forpurification using ANTI-FLAG antibodies, resins, and plates. Othervectors and expression systems are well known in the art for use with avariety of host cells.

In another embodiment two or more peptides or peptide variants of theinvention are encoded and thus expressed in a successive order (similarto “beads on a string” constructs). In doing so, the peptides or peptidevariants may be linked or fused together by stretches of linker aminoacids, such as for example LLLLLL, or may be linked without anyadditional peptide(s) between them. These constructs can also be usedfor cancer therapy, and may induce immune responses both involving MHC Iand MHC II.

The present invention also relates to a host cell transformed with apolynucleotide vector construct of the present invention. The host cellcan be either prokaryotic or eukaryotic. Bacterial cells may bepreferred prokaryotic host cells in some circumstances and typically area strain of E. coli such as, for example, the E. coli strains DH5available from Bethesda Research Laboratories Inc., Bethesda, Md., USA,and RR1 available from the American Type Culture Collection (ATCC) ofRockville, Md., USA (No ATCC 31343). Preferred eukaryotic host cellsinclude yeast, insect and mammalian cells, preferably vertebrate cellssuch as those from a mouse, rat, monkey or human fibroblastic and coloncell lines. Yeast host cells include YPH499, YPH500 and YPH501, whichare generally available from Stratagene Cloning Systems, La Jolla,Calif. 92037, USA. Preferred mammalian host cells include Chinesehamster ovary (CHO) cells available from the ATCC as CCL61, NIH Swissmouse embryo cells NIH/3T3 available from the ATCC as CRL 1658, monkeykidney-derived COS-1 cells available from the ATCC as CRL 1650 and 293cells which are human embryonic kidney cells. Preferred insect cells areSf9 cells which can be transfected with baculovirus expression vectors.An overview regarding the choice of suitable host cells for expressioncan be found in, for example, the textbook of Paulina Balbás and ArgeliaLorence “Methods in Molecular Biology Recombinant Gene Expression,Reviews and Protocols,” Part One, Second Edition, ISBN978-1-58829-262-9, and other literature known to the person of skill.

Transformation of appropriate cell hosts with a DNA construct of thepresent invention is accomplished by well-known methods that typicallydepend on the type of vector used. With regard to transformation ofprokaryotic host cells, see, for example, Cohen et al. (Cohen et al.,1972) and (Green and Sambrook, 2012). Transformation of yeast cells isdescribed in Sherman et al. (Sherman et al., 1986). The method of Beggs(Beggs, 1978) is also useful. With regard to vertebrate cells, reagentsuseful in transfecting such cells, for example calcium phosphate andDEAE-dextran or liposome formulations, are available from StratageneCloning Systems, or Life Technologies Inc., Gaithersburg, Md. 20877,USA. Electroporation is also useful for transforming and/or transfectingcells and is well known in the art for transforming yeast cell,bacterial cells, insect cells and vertebrate cells.

Successfully transformed cells, i.e. cells that contain a DNA constructof the present invention, can be identified by well-known techniquessuch as PCR. Alternatively, the presence of the protein in thesupernatant can be detected using antibodies.

It will be appreciated that certain host cells of the invention areuseful in the preparation of the peptides of the invention, for examplebacterial, yeast and insect cells. However, other host cells may beuseful in certain therapeutic methods. For example, antigen-presentingcells, such as dendritic cells, may usefully be used to express thepeptides of the invention such that they may be loaded into appropriateMHC molecules. Thus, the current invention provides a host cellcomprising a nucleic acid or an expression vector according to theinvention.

In a preferred embodiment, the host cell is an antigen presenting cell,in particular a dendritic cell or antigen presenting cell. APCs loadedwith a recombinant fusion protein containing prostatic acid phosphatase(PAP) were approved by the U.S. Food and Drug Administration (FDA) onApr. 29, 2010, to treat asymptomatic or minimally symptomatic metastaticHRPC (Sipuleucel-T) (Rini et al., 2006; Small et al., 2006).

A further aspect of the invention provides a method of producing apeptide or its variant, the method comprising culturing a host cell andisolating the peptide from the host cell or its culture medium.

In another embodiment, the peptide, the nucleic acid or the expressionvector of the invention are used in medicine. For example, the peptideor its variant may be prepared for intravenous (i.v.) injection,sub-cutaneous (s.c.) injection, intradermal (i.d.) injection,intraperitoneal (i.p.) injection, intramuscular (i.m.) injection.Preferred methods of peptide injection include s.c., i.d., i.p., i.m.,and i.v. Preferred methods of DNA injection include i.d., i.m., s.c.,i.p. and i.v. Doses of e.g. between 50 μg and 1.5 mg, preferably 125 μgto 500 μg, of peptide or DNA may be given and will depend on therespective peptide or DNA. Dosages of this range were successfully usedin previous trials (Walter et al., 2012).

The polynucleotide used for active vaccination may be substantiallypure, or contained in a suitable vector or delivery system. The nucleicacid may be DNA, cDNA, PNA, RNA or a combination thereof. Methods fordesigning and introducing such a nucleic acid are well known in the art.An overview is provided by e.g. Teufel et al. (Teufel et al., 2005).Polynucleotide vaccines are easy to prepare, but the mode of action ofthese vectors in inducing an immune response is not fully understood.Suitable vectors and delivery systems include viral DNA and/or RNA, suchas systems based on adenovirus, vaccinia virus, retroviruses, herpesvirus, adeno-associated virus or hybrids containing elements of morethan one virus. Non-viral delivery systems include cationic lipids andcationic polymers and are well known in the art of DNA delivery.Physical delivery, such as via a “gene-gun” may also be used. Thepeptide or peptides encoded by the nucleic acid may be a fusion protein,for example with an epitope that stimulates T cells for the respectiveopposite CDR as noted above.

The medicament of the invention may also include one or more adjuvants.Adjuvants are substances that non-specifically enhance or potentiate theimmune response (e.g., immune responses mediated by CD8-positive T cellsand helper-T (TH) cells to an antigen, and would thus be considereduseful in the medicament of the present invention. Suitable adjuvantsinclude, but are not limited to, 1018 ISS, aluminum salts, AMPLIVAX®,AS15, BCG, CP-870,893, CpG7909, CyaA, dSLIM, flagellin or TLR5 ligandsderived from flagellin, FLT3 ligand, GM-CSF, IC30, IC31, Imiquimod(ALDARA®), resiquimod, ImuFact IMP321, Interleukins as IL-2, IL-13,IL-21, Interferon-alpha or -beta, or pegylated derivatives thereof, ISPatch, ISS, ISCOMATRIX, ISCOMs, Juvlmmune®, LipoVac, MALP2, MF59,monophosphoryl lipid A, Montanide IMS 1312, Montanide ISA 206, MontanideISA 50V, Montanide ISA-51, water-in-oil and oil-in-water emulsions,OK-432, OM-174, OM-197-MP-EC, ONTAK, OspA, PepTel® vector system,poly(lactid co-glycolid) [PLG]-based and dextran microparticles,talactoferrin SRL172, Virosomes and other Virus-like particles, YF-17D,VEGF trap, R848, beta-glucan, Pam3Cys, Aquila's QS21 stimulon, which isderived from saponin, mycobacterial extracts and synthetic bacterialcell wall mimics, and other proprietary adjuvants such as Ribi's Detox,Quil, or Superfos. Adjuvants such as Freund's or GM-CSF are preferred.Several immunological adjuvants (e.g., MF59) specific for dendriticcells and their preparation have been described previously (Allison andKrummel, 1995). Also, cytokines may be used. Several cytokines have beendirectly linked to influencing dendritic cell migration to lymphoidtissues (e.g., TNF-), accelerating the maturation of dendritic cellsinto efficient antigen-presenting cells for T-lymphocytes (e.g., GM-CSF,IL-1 and IL-4) (U.S. Pat. No. 5,849,589, specifically incorporatedherein by reference in its entirety) and acting as immunoadjuvants(e.g., IL-12, IL-15, IL-23, IL-7, IFN-alpha. IFN-beta) (Gabrilovich etal., 1996).

CpG immunostimulatory oligonucleotides have also been reported toenhance the effects of adjuvants in a vaccine setting. Without beingbound by theory, CpG oligonucleotides act by activating the innate(non-adaptive) immune system via Toll-like receptors (TLR), mainly TLR9.CpG triggered TLR9 activation enhances antigen-specific humoral andcellular responses to a wide variety of antigens, including peptide orprotein antigens, live or killed viruses, dendritic cell vaccines,autologous cellular vaccines and polysaccharide conjugates in bothprophylactic and therapeutic vaccines. More importantly it enhancesdendritic cell maturation and differentiation, resulting in enhancedactivation of TH1 cells and strong cytotoxic T-lymphocyte (CTL)generation, even in the absence of CD4 T cell help. The TH1 bias inducedby TLR9 stimulation is maintained even in the presence of vaccineadjuvants such as alum or incomplete Freund's adjuvant (IFA) thatnormally promote a TH2 bias. CpG oligonucleotides show even greateradjuvant activity when formulated or co-administered with otheradjuvants or in formulations such as microparticles, nanoparticles,lipid emulsions or similar formulations, which are especially necessaryfor inducing a strong response when the antigen is relatively weak. Theyalso accelerate the immune response and enable the antigen doses to bereduced by approximately two orders of magnitude, with comparableantibody responses to the full-dose vaccine without CpG in someexperiments (Krieg, 2006). U.S. Pat. No. 6,406,705 B1 describes thecombined use of CpG oligonucleotides, non-nucleic acid adjuvants and anantigen to induce an antigen-specific immune response. A CpG TLR9antagonist is dSLIM (double Stem Loop Immunomodulator) by Mologen(Berlin, Germany) which is a preferred component of the pharmaceuticalcomposition of the present invention. Other TLR binding molecules suchas RNA binding TLR 7, TLR 8 and/or TLR 9 may also be used.

Other examples for useful adjuvants include, but are not limited tochemically modified CpGs (e.g. CpR, Idera), dsRNA analogues such asPoly(I:C) and derivates thereof (e.g. AmpliGen®, Hiltonol®, poly-(ICLC),poly(IC-R), poly(I:C12U), non-CpG bacterial DNA or RNA as well asimmunoactive small molecules and antibodies such as cyclophosphamide,sunitinib, Bevacizumab®, celebrex, NCX-4016, sildenafil, tadalafil,vardenafil, sorafenib, temozolomide, temsirolimus, XL-999, CP-547632,pazopanib, VEGF Trap, ZD2171, AZD2171, anti-CTLA4, other antibodiestargeting key structures of the immune system (e.g. anti-CD40,anti-TGFbeta, anti-TNFalpha receptor) and SC58175, which may acttherapeutically and/or as an adjuvant. The amounts and concentrations ofadjuvants and additives useful in the context of the present inventioncan readily be determined by the skilled artisan without undueexperimentation.

Preferred adjuvants are anti-CD40, imiquimod, resiquimod, GM-CSF,cyclophosphamide, sunitinib, bevacizumab, interferon-alpha, CpGoligonucleotides and derivates, poly-(I:C) and derivates, RNA,sildenafil, and particulate formulations with PLG or virosomes.

In a preferred embodiment, the pharmaceutical composition according tothe invention the adjuvant is selected from the group consisting ofcolony-stimulating factors, such as Granulocyte Macrophage ColonyStimulating Factor (GM-CSF, sargramostim), cyclophosphamide, imiquimod,resiquimod, and interferon-alpha.

In a preferred embodiment, the pharmaceutical composition according tothe invention the adjuvant is selected from the group consisting ofcolony-stimulating factors, such as Granulocyte Macrophage ColonyStimulating Factor (GM-CSF, sargramostim), cyclophosphamide, imiquimodand resiquimod. In a preferred embodiment of the pharmaceuticalcomposition according to the invention, the adjuvant iscyclophosphamide, imiquimod or resiquimod. Even more preferred adjuvantsare Montanide IMS 1312, Montanide ISA 206, Montanide ISA 50V, MontanideISA-51, poly-ICLC (Hiltonol®) and anti-CD40 mAB, or combinationsthereof.

This composition is used for parenteral administration, such assubcutaneous, intradermal, intramuscular or oral administration. Forthis, the peptides and optionally other molecules are dissolved orsuspended in a pharmaceutically acceptable, preferably aqueous carrier.In addition, the composition can contain excipients, such as buffers,binding agents, blasting agents, diluents, flavors, lubricants, etc. Thepeptides can also be administered together with immune stimulatingsubstances, such as cytokines. An extensive listing of excipients thatcan be used in such a composition, can be, for example, taken from A.Kibbe, Handbook of Pharmaceutical Excipients (Kibbe, 2000). Thecomposition can be used for a prevention, prophylaxis and/or therapy ofadenomatous or cancerous diseases. Exemplary formulations can be foundin, for example, EP2112253.

In an aspect, peptides or other molecules described herein may becombined with an aquous carrier. In an aspect, the aquous carrier isselected from ion exchangers, alumina, aluminum stearate, magnesiumstearate, lecithin, serum proteins, such as human serum albumin, buffersubstances such as phosphates, glycine, sorbic acid, potassium sorbate,partial glyceride mixtures of saturated vegetable fatty acids, salts orelectrolytes, such as protamine sulfate, disodium hydrogen phosphate,dicalcium phosphate, potassium hydrogen phosphate, sodium chloride, zincsalts, colloidal silica, magnesium trisilicate, polyvinyl pyrrolidone,polyvinylpyrrolidone-vinyl acetate, cellulose-based substances (e.g.,microcrystalline cellulose, hydroxypropyl methylcellulose, hydroxypropylmethylcellulose acetate succinate, hydroxypropyl methylcellulosePhthalate), starch, lactose monohydrate, mannitol, trehalose sodiumlauryl sulfate, and crosscarmellose sodium, polyethylene glycol, sodiumcarboxymethylcellulose, polyacrylates, polymethacrylate, waxes,polyethylene-polyoxypropylene-block polymers, polyethylene glycol andwool fat.

In an aspect, the aquous carrier contains multiple components, such aswater together with a non-water carrier component, such as thosecomponents described herein. In another aspect, the aquous carrier iscapable of imparting improved properties when combined with a peptide orother molecule described herein, for example, improved solubility,efficiacy, and/or improved immunotherapy. In addition, the compositioncan contain excipients, such as buffers, binding agents, blastingagents, diluents, flavors, lubricants, etc. A “pharmaceuticallyacceptable diluent,” for example, may include solvents, bulking agents,stabilizing agents, dispersion media, coatings, antibacterial andantifungal agents, isotonic and absorption delaying agents, and the likewhich are physiologically compatible. Examples of pharmaceuticallyacceptable diluents include one or more of saline, phosphate bufferedsaline, dextrose, glycerol, ethanol, and the like as well ascombinations thereof. In many cases it will be preferable to include oneor more isotonic agents, for example, sugars such as trehalose andsucrose, polyalcohols such as mannitol, sorbitol, or sodium chloride inthe composition. Pharmaceutically acceptable substances such as wettingor minor amounts of auxiliary substances such as wetting or emulsifyingagents, preservatives or buffers, are also within the scope of thepresent invention. In addition, the composition can contain excipients,such as buffers, binding agents, blasting agents, diluents, flavors, andlubricants.

It is important to realize that the immune response triggered by thevaccine according to the invention attacks the cancer in differentcell-stages and different stages of development. Furthermore, differentcancer associated signaling pathways are attacked. This is an advantageover vaccines that address only one or few targets, which may cause thetumor to easily adapt to the attack (tumor escape). Furthermore, not allindividual tumors express the same pattern of antigens. Therefore, acombination of several tumor-associated peptides ensures that everysingle tumor bears at least some of the targets. The composition isdesigned in such a way that each tumor is expected to express several ofthe antigens and cover several independent pathways necessary for tumorgrowth and maintenance. Thus, the vaccine can easily be used“off-the-shelf” for a larger patient population. This means that apre-selection of patients to be treated with the vaccine can berestricted to HLA typing, does not require any additional biomarkerassessments for antigen expression, but it is still ensured that severaltargets are simultaneously attacked by the induced immune response,which is important for efficacy (Banchereau et al., 2001; Walter et al.,2012).

As used herein, the term “scaffold” refers to a molecule thatspecifically binds to an (e.g. antigenic) determinant. In oneembodiment, a scaffold is able to direct the entity to which it isattached (e.g. a (second) antigen binding moiety) to a target site, forexample to a specific type of tumor cell or tumor stroma bearing theantigenic determinant (e.g. the complex of a peptide with MHC, accordingto the application at hand). In another embodiment, a scaffold is ableto activate signaling through its target antigen, for example a T cellreceptor complex antigen. Scaffolds include but are not limited toantibodies and fragments thereof, antigen binding domains of anantibody, comprising an antibody heavy chain variable region and anantibody light chain variable region, binding proteins comprising atleast one ankyrin repeat motif and single domain antigen binding (SDAB)molecules, aptamers, (soluble) TCRs and (modified) cells such asallogenic or autologous T cells. To assess whether a molecule is ascaffold binding to a target, binding assays can be performed.

“Specific” binding means that the scaffold binds the peptide-MHC-complexof interest better than other naturally occurring peptide-MHC-complexes,to an extent that a scaffold armed with an active molecule that is ableto kill a cell bearing the specific target is not able to kill anothercell without the specific target but presenting other peptide-MHCcomplex(es). Binding to other peptide-MHC complexes is irrelevant if thepeptide of the cross-reactive peptide-MHC is not naturally occurring,i.e. not derived from the human HLA-peptidome. Tests to assess targetcell killing are well known in the art. They should be performed usingtarget cells (primary cells or cell lines) with unaltered peptide-MHCpresentation, or cells loaded with peptides such that naturallyoccurring peptide-MHC levels are reached.

Each scaffold can comprise a labelling which provides that the boundscaffold can be detected by determining the presence or absence of asignal provided by the label. For example, the scaffold can be labelledwith a fluorescent dye or any other applicable cellular marker molecule.Such marker molecules are well known in the art. For example, afluorescence-labelling, for example provided by a fluorescence dye, canprovide a visualization of the bound aptamer by fluorescence or laserscanning microscopy or flow cytometry.

Each scaffold can be conjugated with a second active molecule such asfor example IL-21, anti-CD3, and anti-CD28.

For further information on polypeptide scaffolds see for example thebackground section of WO 2014/071978A1 and the references cited therein.

The present invention further relates to aptamers. Aptamers (see forexample WO 2014/191359 and the literature as cited therein) are shortsingle-stranded nucleic acid molecules, which can fold into definedthree-dimensional structures and recognize specific target structures.They have appeared to be suitable alternatives for developing targetedtherapies. Aptamers have been shown to selectively bind to a variety ofcomplex targets with high affinity and specificity.

Aptamers recognizing cell surface located molecules have been identifiedwithin the past decade and provide means for developing diagnostic andtherapeutic approaches. Since aptamers have been shown to possess almostno toxicity and immunogenicity they are promising candidates forbiomedical applications. Indeed aptamers, for example prostate-specificmembrane-antigen recognizing aptamers, have been successfully employedfor targeted therapies and shown to be functional in xenograft in vivomodels. Furthermore, aptamers recognizing specific tumor cell lines havebeen identified.

DNA aptamers can be selected to reveal broad-spectrum recognitionproperties for various cancer cells, and particularly those derived fromsolid tumors, while non-tumorigenic and primary healthy cells are notrecognized. If the identified aptamers recognize not only a specifictumor sub-type but rather interact with a series of tumors, this rendersthe aptamers applicable as so-called broad-spectrum diagnostics andtherapeutics.

Further, investigation of cell-binding behavior with flow cytometryshowed that the aptamers revealed very good apparent affinities that arewithin the nanomolar range.

Aptamers are useful for diagnostic and therapeutic purposes. Further, itcould be shown that some of the aptamers are taken up by tumor cells andthus can function as molecular vehicles for the targeted delivery ofanti-cancer agents such as siRNA into tumor cells.

Aptamers can be selected against complex targets such as cells andtissues and complexes of the peptides comprising, preferably consistingof, a sequence according to any of SEQ ID NO 1 to SEQ ID NO 489,according to the present invention with the MHC molecule, using thecell-SELEX (Systematic Evolution of Ligands by Exponential enrichment)technique.

The peptides of the present invention can be used to generate anddevelop specific antibodies against MHC/peptide complexes. These can beused for therapy, targeting toxins or radioactive substances to thediseased tissue. Another use of these antibodies can be targetingradionuclides to the diseased tissue for imaging purposes such as PET.This use can help to detect small metastases or to determine the sizeand precise localization of diseased tissues.

Therefore, it is a further aspect of the invention to provide a methodfor producing a recombinant antibody specifically binding to a humanmajor histocompatibility complex (MHC) class I or II being complexedwith a HLA-restricted antigen (preferably a peptide according to thepresent invention), the method comprising: immunizing a geneticallyengineered non-human mammal comprising cells expressing said human majorhistocompatibility complex (MHC) class I or II with a soluble form of aMHC class I or II molecule being complexed with said HLA-restrictedantigen; isolating mRNA molecules from antibody producing cells of saidnon-human mammal; producing a phage display library displaying proteinmolecules encoded by said mRNA molecules; and isolating at least onephage from said phage display library, said at least one phagedisplaying said antibody specifically binding to said human majorhistocompatibility complex (MHC) class I or II being complexed with saidHLA-restricted antigen.

It is thus a further aspect of the invention to provide an antibody thatspecifically binds to a human major histocompatibility complex (MHC)class I or II being complexed with a HLA-restricted antigen, wherein theantibody preferably is a polyclonal antibody, monoclonal antibody,bi-specific antibody and/or a chimeric antibody.

Respective methods for producing such antibodies and single chain classI major histocompatibility complexes, as well as other tools for theproduction of these antibodies are disclosed in WO 03/068201, WO2004/084798, WO 01/72768, WO 03/070752, and in publications (Cohen etal., 2003a; Cohen et al., 2003b; Denkberg et al., 2003), which for thepurposes of the present invention are all explicitly incorporated byreference in their entireties.

Preferably, the antibody is binding with a binding affinity of below 20nanomolar, preferably of below 10 nanomolar, to the complex, which isalso regarded as “specific” in the context of the present invention.

The present invention relates to a peptide comprising a sequence that isselected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 489, ora variant thereof which is at least 88% homologous (preferablyidentical) to SEQ ID NO: 1 to SEQ ID NO: 489 or a variant thereof thatinduces T cells cross-reacting with said peptide, wherein said peptideis not the underlying full-length polypeptide.

The present invention further relates to a peptide comprising a sequencethat is selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO:489 or a variant thereof which is at least 88% homologous (preferablyidentical) to SEQ ID NO: 1 to SEQ ID NO: 489, wherein said peptide orvariant has an overall length of between 8 and 100, preferably between 8and 30, and most preferred between 8 and 14 amino acids.

The present invention further relates to the peptides according to theinvention that have the ability to bind to a molecule of the human majorhistocompatibility complex (MHC) class-I or -II.

The present invention further relates to the peptides according to theinvention wherein the peptide consists or consists essentially of anamino acid sequence according to SEQ ID NO: 1 to SEQ ID NO: 489.

The present invention further relates to the peptides according to theinvention, wherein the peptide is (chemically) modified and/or includesnon-peptide bonds.

The present invention further relates to the peptides according to theinvention, wherein the peptide is part of a fusion protein, inparticular comprising N-terminal amino acids of the HLA-DRantigen-associated invariant chain (li), or wherein the peptide is fusedto (or into) an antibody, such as, for example, an antibody that isspecific for dendritic cells.

The present invention further relates to a nucleic acid, encoding thepeptides according to the invention, provided that the peptide is notthe complete (full) human protein.

The present invention further relates to the nucleic acid according tothe invention that is DNA, cDNA, PNA, RNA or combinations thereof.

The present invention further relates to an expression vector capable ofexpressing a nucleic acid according to the present invention.

The present invention further relates to a peptide according to thepresent invention, a nucleic acid according to the present invention oran expression vector according to the present invention for use inmedicine, in particular in the treatment of lung cancer (including NSCLCand SCLC).

The present invention further relates to a host cell comprising anucleic acid according to the present invention or an expression vectoraccording to the present invention.

The present invention further relates to the host cell according to thepresent invention that is an antigen presenting cell, and preferably adendritic cell.

The present invention further relates to a method of producing a peptideaccording to the present invention, said method comprising culturing thehost cell according to the present invention, and isolating the peptidefrom said host cell or its culture medium.

The present invention further relates to the method according to thepresent invention, where-in the antigen is loaded onto class I or II MHCmolecules expressed on the surface of a suitable antigen-presenting cellby contacting a sufficient amount of the antigen with anantigen-presenting cell.

The present invention further relates to the method according to theinvention, wherein the antigen-presenting cell comprises an expressionvector capable of expressing said peptide containing SEQ ID NO: 1 to SEQID NO: 489 or said variant amino acid sequence.

The present invention further relates to activated T cells, produced bythe method according to the present invention, wherein said T cellsselectively recognizes a cell which aberrantly expresses a polypeptidecomprising an amino acid sequence according to the present invention.

The present invention further relates to a method of killing targetcells in a patient which target cells aberrantly express a polypeptidecomprising any amino acid sequence according to the present invention,the method comprising administering to the patient an effective numberof T cells as according to the present invention.

The present invention further relates to the use of any peptidedescribed, a nucleic acid according to the present invention, anexpression vector according to the present invention, a cell accordingto the present invention, or an activated cytotoxic T lymphocyteaccording to the present invention as a medicament or in the manufactureof a medicament. The present invention further relates to a useaccording to the present invention, wherein the medicament is activeagainst cancer.

The present invention further relates to a use according to theinvention, wherein the medicament is a vaccine. The present inventionfurther relates to a use according to the invention, wherein themedicament is active against cancer.

The present invention further relates to a use according to theinvention, wherein said cancer cells are lung cancer (including NSCLCand SCLC) cells or other solid or hematological tumor cells such asacute myeloid leukemia, breast cancer, bile duct cancer, brain cancer,chronic lymphocytic leukemia, colorectal carcinoma, esophageal cancer,gallbladder cancer, gastric cancer, head and neck squamous cellcarcinoma, hepatocellular cancer, melanoma, non-Hodgkin lymphoma,ovarian cancer, pancreatic cancer, prostate cancer, renal cell cancer,urinary bladder cancer, uterine cancer.

The present invention further relates to particular marker proteins andbiomarkers based on the peptides according to the present invention,herein called “targets” that can be used in the diagnosis and/orprognosis of lung cancer (including NSCLC and SCLC). The presentinvention also relates to the use of these novel targets for cancertreatment.

The term “antibody” or “antibodies” is used herein in a broad sense andincludes both polyclonal and monoclonal antibodies. In addition tointact or “full” immunoglobulin molecules, also included in the term“antibodies” are fragments (e.g. CDRs, Fv, Fab and Fc fragments) orpolymers of those immunoglobulin molecules and humanized versions ofimmunoglobulin molecules, as long as they exhibit any of the desiredproperties (e.g., specific binding of a lung cancer (including NSCLC andSCLC) marker (poly)peptide, delivery of a toxin to a lung cancer(including NSCLC and SCLC) cell expressing a cancer marker gene at anincreased level, and/or inhibiting the activity of a lung cancer(including NSCLC and SCLC) marker polypeptide) according to theinvention.

Whenever possible, the antibodies of the invention may be purchased fromcommercial sources. The antibodies of the invention may also begenerated using well-known methods. The skilled artisan will understandthat either full length lung cancer (including NSCLC and SCLC) markerpolypeptides or fragments thereof may be used to generate the antibodiesof the invention. A polypeptide to be used for generating an antibody ofthe invention may be partially or fully purified from a natural source,or may be produced using recombinant DNA techniques.

For example, a cDNA encoding a peptide according to the presentinvention, such as a peptide according to SEQ ID NO: 1 to SEQ ID NO: 489polypeptide, or a variant or fragment thereof, can be expressed inprokaryotic cells (e.g., bacteria) or eukaryotic cells (e.g., yeast,insect, or mammalian cells), after which the recombinant protein can bepurified and used to generate a monoclonal or polyclonal antibodypreparation that specifically bind the lung cancer (including NSCLC andSCLC) marker polypeptide used to generate the antibody according to theinvention.

One of skill in the art will realize that the generation of two or moredifferent sets of monoclonal or polyclonal antibodies maximizes thelikelihood of obtaining an antibody with the specificity and affinityrequired for its intended use (e.g., ELISA, immunohistochemistry, invivo imaging, immunotoxin therapy). The antibodies are tested for theirdesired activity by known methods, in accordance with the purpose forwhich the antibodies are to be used (e.g., ELISA, immunohistochemistry,immunotherapy, etc.; for further guidance on the generation and testingof antibodies, see, e.g., Greenfield, 2014 (Greenfield, 2014)). Forexample, the antibodies may be tested in ELISA assays or, Western blots,immunohistochemical staining of formalin-fixed cancers or frozen tissuesections. After their initial in vitro characterization, antibodiesintended for therapeutic or in vivo diagnostic use are tested accordingto known clinical testing methods.

The term “monoclonal antibody” as used herein refers to an antibodyobtained from a substantially homogeneous population of antibodies,i.e.; the individual antibodies comprising the population are identicalexcept for possible naturally occurring mutations that may be present inminor amounts. The monoclonal antibodies herein specifically include“chimeric” antibodies in which a portion of the heavy and/or light chainis identical with or homologous to corresponding sequences in antibodiesderived from a particular species or belonging to a particular antibodyclass or subclass, while the remainder of the chain(s) is identical withor homologous to corresponding sequences in antibodies derived fromanother species or belonging to another antibody class or subclass, aswell as fragments of such antibodies, so long as they exhibit thedesired antagonistic activity (U.S. Pat. No. 4,816,567, which is herebyincorporated in its entirety).

Monoclonal antibodies of the invention may be prepared using hybridomamethods. In a hybridoma method, a mouse or other appropriate host animalis typically immunized with an immunizing agent to elicit lymphocytesthat produce or are capable of producing antibodies that willspecifically bind to the immunizing agent. Alternatively, thelymphocytes may be immunized in vitro.

The monoclonal antibodies may also be made by recombinant DNA methods,such as those described in U.S. Pat. No. 4,816,567. DNA encoding themonoclonal antibodies of the invention can be readily isolated andsequenced using conventional procedures (e.g., by using oligonucleotideprobes that are capable of binding specifically to genes encoding theheavy and light chains of murine antibodies).

In vitro methods are also suitable for preparing monovalent antibodies.Digestion of antibodies to produce fragments thereof, particularly Fabfragments, can be accomplished using routine techniques known in theart. For instance, digestion can be performed using papain. Examples ofpapain digestion are described in WO 94/29348 and U.S. Pat. No.4,342,566. Papain digestion of antibodies typically produces twoidentical antigen binding fragments, called Fab fragments, each with asingle antigen binding site, and a residual Fc fragment. Pepsintreatment yields a F(ab′)2 fragment and a pFc′ fragment.

The antibody fragments, whether attached to other sequences or not, canalso include insertions, deletions, substitutions, or other selectedmodifications of particular regions or specific amino acids residues,provided the activity of the fragment is not significantly altered orimpaired compared to the non-modified antibody or antibody fragment.These modifications can provide for some additional property, such as toremove/add amino acids capable of disulfide bonding, to increase itsbio-longevity, to alter its secretory characteristics, etc. In any case,the antibody fragment must possess a bioactive property, such as bindingactivity, regulation of binding at the binding domain, etc. Functionalor active regions of the antibody may be identified by mutagenesis of aspecific region of the protein, followed by expression and testing ofthe expressed polypeptide. Such methods are readily apparent to askilled practitioner in the art and can include site-specificmutagenesis of the nucleic acid encoding the antibody fragment.

The antibodies of the invention may further comprise humanizedantibodies or human antibodies. Humanized forms of non-human (e.g.,murine) antibodies are chimeric immunoglobulins, immunoglobulin chainsor fragments thereof (such as Fv, Fab, Fab′ or other antigen-bindingsubsequences of antibodies) which contain minimal sequence derived fromnon-human immunoglobulin. Humanized antibodies include humanimmunoglobulins (recipient antibody) in which residues from acomplementary determining region (CDR) of the recipient are replaced byresidues from a CDR of a non-human species (donor antibody) such asmouse, rat or rabbit having the desired specificity, affinity andcapacity. In some instances, Fv framework (FR) residues of the humanimmunoglobulin are replaced by corresponding non-human residues.Humanized antibodies may also comprise residues which are found neitherin the recipient antibody nor in the imported CDR or frameworksequences. In general, the humanized antibody will comprisesubstantially all of at least one, and typically two, variable domains,in which all or substantially all of the CDR regions correspond to thoseof a non-human immunoglobulin and all or substantially all of the FRregions are those of a human immunoglobulin consensus sequence. Thehumanized antibody optimally also will comprise at least a portion of animmunoglobulin constant region (Fc), typically that of a humanimmunoglobulin.

Methods for humanizing non-human antibodies are well known in the art.Generally, a humanized antibody has one or more amino acid residuesintroduced into it from a source which is non-human. These non-humanamino acid residues are often referred to as “import” residues, whichare typically taken from an “import” variable domain. Humanization canbe essentially performed by substituting rodent CDRs or CDR sequencesfor the corresponding sequences of a human antibody. Accordingly, such“humanized” antibodies are chimeric antibodies (U.S. Pat. No.4,816,567), wherein substantially less than an intact human variabledomain has been substituted by the corresponding sequence from anon-human species. In practice, humanized antibodies are typically humanantibodies in which some CDR residues and possibly some FR residues aresubstituted by residues from analogous sites in rodent antibodies.

Transgenic animals (e.g., mice) that are capable, upon immunization, ofproducing a full repertoire of human antibodies in the absence ofendogenous immunoglobulin production can be employed. For example, ithas been described that the homozygous deletion of the antibody heavychain joining region gene in chimeric and germ-line mutant mice resultsin complete inhibition of endogenous antibody production. Transfer ofthe human germ-line immunoglobulin gene array in such germ-line mutantmice will result in the production of human antibodies upon antigenchallenge. Human antibodies can also be produced in phage displaylibraries.

Antibodies of the invention are preferably administered to a subject ina pharmaceutically acceptable carrier. Typically, an appropriate amountof a pharmaceutically-acceptable salt is used in the formulation torender the formulation isotonic. Examples of thepharmaceutically-acceptable carrier include saline, Ringer's solutionand dextrose solution. The pH of the solution is preferably from about 5to about 8, and more preferably from about 7 to about 7.5. Furthercarriers include sustained release preparations such as semipermeablematrices of solid hydrophobic polymers containing the antibody, whichmatrices are in the form of shaped articles, e.g., films, liposomes ormicroparticles. It will be apparent to those persons skilled in the artthat certain carriers may be more preferable depending upon, forinstance, the route of administration and concentration of antibodybeing administered.

The antibodies can be administered to the subject, patient, or cell byinjection (e.g., intravenous, intraperitoneal, subcutaneous,intramuscular), or by other methods such as infusion that ensure itsdelivery to the bloodstream in an effective form. The antibodies mayalso be administered by intratumoral or peritumoral routes, to exertlocal as well as systemic therapeutic effects. Local or intravenousinjection is preferred.

Effective dosages and schedules for administering the antibodies may bedetermined empirically, and making such determinations is within theskill in the art. Those skilled in the art will understand that thedosage of antibodies that must be administered will vary depending on,for example, the subject that will receive the antibody, the route ofadministration, the particular type of antibody used and other drugsbeing administered. A typical daily dosage of the antibody used alonemight range from about 1 (μg/kg to up to 100 mg/kg of body weight ormore per day, depending on the factors mentioned above. Followingadministration of an antibody, preferably for treating lung cancer(including NSCLC and SCLC), the efficacy of the therapeutic antibody canbe assessed in various ways well known to the skilled practitioner. Forinstance, the size, number, and/or distribution of cancer in a subjectreceiving treatment may be monitored using standard tumor imagingtechniques. A therapeutically-administered antibody that arrests tumorgrowth, results in tumor shrinkage, and/or prevents the development ofnew tumors, compared to the disease course that would occurs in theabsence of antibody administration, is an efficacious antibody fortreatment of cancer.

It is a further aspect of the invention to provide a method forproducing a soluble T-cell receptor (sTCR) recognizing a specificpeptide-MHC complex. Such soluble T-cell receptors can be generated fromspecific T-cell clones, and their affinity can be increased bymutagenesis targeting the complementarity-determining regions. For thepurpose of T-cell receptor selection, phage display can be used (US2010/0113300, (Liddy et al., 2012)). For the purpose of stabilization ofT-cell receptors during phage display and in case of practical use asdrug, alpha and beta chain can be linked e.g. by non-native disulfidebonds, other covalent bonds (single-chain T-cell receptor), or bydimerization domains (Boulter et al., 2003; Card et al., 2004; Willcoxet al., 1999). The T-cell receptor can be linked to toxins, drugs,cytokines (see, for example, US 2013/0115191), and domains recruitingeffector cells such as an anti-CD3 domain, etc., in order to executeparticular functions on target cells. Moreover, it could be expressed inT cells used for adoptive transfer. Further information can be found inWO 2004/033685A1 and WO 2004/074322A1. A combination of sTCRs isdescribed in WO 2012/056407A1. Further methods for the production aredisclosed in WO 2013/057586A1.

In addition, the peptides and/or the TCRs or antibodies or other bindingmolecules of the present invention can be used to verify a pathologist'sdiagnosis of a cancer based on a biopsied sample.

The antibodies or TCRs may also be used for in vivo diagnostic assays.Generally, the antibody is labeled with a radionucleotide (such as¹¹¹In, ⁹⁹Tc, ¹⁴C, ¹³¹I, ³H, ³²P or ³⁵S) so that the tumor can belocalized using immunoscintiography. In one embodiment, antibodies orfragments thereof bind to the extracellular domains of two or moretargets of a protein selected from the group consisting of theabove-mentioned proteins, and the affinity value (Kd) is less than 1×10μM.

Antibodies for diagnostic use may be labeled with probes suitable fordetection by various imaging methods. Methods for detection of probesinclude, but are not limited to, fluorescence, light, confocal andelectron microscopy; magnetic resonance imaging and spectroscopy;fluoroscopy, computed tomography and positron emission tomography.Suitable probes include, but are not limited to, fluorescein, rhodamine,eosin and other fluorophores, radioisotopes, gold, gadolinium and otherlanthanides, paramagnetic iron, fluorine-18 and other positron-emittingradionuclides. Additionally, probes may be bi- or multi-functional andbe detectable by more than one of the methods listed. These antibodiesmay be directly or indirectly labeled with said probes. Attachment ofprobes to the antibodies includes covalent attachment of the probe,incorporation of the probe into the antibody, and the covalentattachment of a chelating compound for binding of probe, amongst otherswell recognized in the art. For immunohistochemistry, the disease tissuesample may be fresh or frozen or may be embedded in paraffin and fixedwith a preservative such as formalin. The fixed or embedded sectioncontains the sample are contacted with a labeled primary antibody andsecondary antibody, wherein the antibody is used to detect theexpression of the proteins in situ.

Another aspect of the present invention includes an in vitro method forproducing activated T cells, the method comprising contacting in vitro Tcells with antigen loaded human MHC molecules expressed on the surfaceof a suitable antigen-presenting cell for a period of time sufficient toactivate the T cell in an antigen specific manner, wherein the antigenis a peptide according to the invention. Preferably a sufficient amountof the antigen is used with an antigen-presenting cell.

Preferably the mammalian cell lacks or has a reduced level or functionof the TAP peptide transporter. Suitable cells that lack the TAP peptidetransporter include T2, RMA-S and Drosophila cells. TAP is thetransporter associated with antigen processing.

The human peptide loading deficient cell line T2 is available from theAmerican Type Culture Collection, 12301 Parklawn Drive, Rockville, Md.20852, USA under Catalogue No CRL 1992; the Drosophila cell lineSchneider line 2 is available from the ATCC under Catalogue No CRL19863; the mouse RMA-S cell line is described in Ljunggren et al.(Ljunggren and Karre, 1985).

Preferably, before transfection the host cell expresses substantially noMHC class I molecules. It is also preferred that the stimulator cellexpresses a molecule important for providing a co-stimulatory signal forT-cells such as any of B7.1, B7.2, ICAM-1 and LFA 3. The nucleic acidsequences of numerous MHC class I molecules and of the co-stimulatormolecules are publicly available from the GenBank and EMBL databases.

In case of a MHC class I epitope being used as an antigen, the T cellsare CD8-positive T cells.

If an antigen-presenting cell is transfected to express such an epitope,preferably the cell comprises an expression vector capable of expressinga peptide containing SEQ ID NO: 1 to SEQ ID NO: 489, or a variant aminoacid sequence thereof.

A number of other methods may be used for generating T cells in vitro.For example, autologous tumor-infiltrating lymphocytes can be used inthe generation of CTL. Plebanski et al. (Plebanski et al., 1995) madeuse of autologous peripheral blood lymphocytes (PLBs) in the preparationof T cells. Furthermore, the production of autologous T cells by pulsingdendritic cells with peptide or polypeptide, or via infection withrecombinant virus is possible. Also, B cells can be used in theproduction of autologous T cells. In addition, macrophages pulsed withpeptide or polypeptide, or infected with recombinant virus, may be usedin the preparation of autologous T cells. S. Walter et al. (Walter etal., 2003) describe the in vitro priming of T cells by using artificialantigen presenting cells (aAPCs), which is also a suitable way forgenerating T cells against the peptide of choice. In the presentinvention, aAPCs were generated by the coupling of preformed MHC:peptidecomplexes to the surface of polystyrene particles (microbeads) bybiotin:streptavidin biochemistry. This system permits the exact controlof the MHC density on aAPCs, which allows to selectively elicit high- orlow-avidity antigen-specific T cell responses with high efficiency fromblood samples. Apart from MHC:peptide complexes, aAPCs should carryother proteins with co-stimulatory activity like anti-CD28 antibodiescoupled to their surface. Furthermore, such aAPC-based systems oftenrequire the addition of appropriate soluble factors, e. g. cytokines,like interleukin-12.

Allogeneic cells may also be used in the preparation of T cells and amethod is described in detail in WO 97/26328, incorporated herein byreference. For example, in addition to Drosophila cells and T2 cells,other cells may be used to present antigens such as CHO cells,baculovirus-infected insect cells, bacteria, yeast, andvaccinia-infected target cells. In addition, plant viruses may be used(see, for example, Porta et al. (Porta et al., 1994) which describes thedevelopment of cowpea mosaic virus as a high-yielding system for thepresentation of foreign peptides.

The activated T cells that are directed against the peptides of theinvention are useful in therapy. Thus, a further aspect of the inventionprovides activated T cells obtainable by the foregoing methods of theinvention.

Activated T cells, which are produced by the above method, willselectively recognize a cell that aberrantly expresses a polypeptidethat comprises an amino acid sequence of SEQ ID NO: 1 to SEQ ID NO 489.

Preferably, the T cell recognizes the cell by interacting through itsTCR with the HLA/peptide-complex (for example, binding). The T cells areuseful in a method of killing target cells in a patient whose targetcells aberrantly express a polypeptide comprising an amino acid sequenceof the invention wherein the patient is administered an effective numberof the activated T cells. The T cells that are administered to thepatient may be derived from the patient and activated as described above(i.e. they are autologous T cells). Alternatively, the T cells are notfrom the patient but are from another individual. Of course, it ispreferred if the individual is a healthy individual. By “healthyindividual” the inventors mean that the individual is generally in goodhealth, preferably has a competent immune system and, more preferably,is not suffering from any disease that can be readily tested for, anddetected.

In vivo, the target cells for the CD8-positive T cells according to thepresent invention can be cells of the tumor (which sometimes express MHCclass II) and/or stromal cells surrounding the tumor (tumor cells)(which sometimes also express MHC class II; (Dengjel et al., 2006)).

The T cells of the present invention may be used as active ingredientsof a therapeutic composition. Thus, the invention also provides a methodof killing target cells in a patient whose target cells aberrantlyexpress a polypeptide comprising an amino acid sequence of theinvention, the method comprising administering to the patient aneffective number of T cells as defined above.

By “aberrantly expressed” the inventors also mean that the polypeptideis over-expressed compared to levels of expression in normal tissues orthat the gene is silent in the tissue from which the tumor is derivedbut in the tumor it is expressed. By “over-expressed” the inventors meanthat the polypeptide is present at a level at least 1.2-fold of thatpresent in normal tissue; preferably at least 2-fold, and morepreferably at least 4-fold or 6-fold the level present in normal tissue.

T cells may be obtained by methods known in the art, e.g. thosedescribed above.

Protocols for this so-called adoptive transfer of T cells are well knownin the art. Reviews can be found in: Gattioni et al. and Morgan et al.(Gattinoni et al., 2006; Morgan et al., 2006).

Another aspect of the present invention includes the use of the peptidescomplexed with MHC to generate a T-cell receptor whose nucleic acid iscloned and is introduced into a host cell, preferably a T cell. Thisengineered T cell can then be transferred to a patient for therapy ofcancer.

Any molecule of the invention, i.e. the peptide, nucleic acid, antibody,expression vector, cell, activated T cell, T-cell receptor or thenucleic acid encoding it, is useful for the treatment of disorders,characterized by cells escaping an immune response. Therefore, anymolecule of the present invention may be used as medicament or in themanufacture of a medicament. The molecule may be used by itself orcombined with other molecule(s) of the invention or (a) knownmolecule(s).

The present invention is further directed at a kit comprising:

(a) a container containing a pharmaceutical composition as describedabove, in solution or in lyophilized form;

(b) optionally a second container containing a diluent or reconstitutingsolution for the lyophilized formulation; and

(c) optionally, instructions for (i) use of the solution or (ii)reconstitution and/or use of the lyophilized formulation.

The kit may further comprise one or more of (iii) a buffer, (iv) adiluent, (v) a filter, (vi) a needle, or (v) a syringe. The container ispreferably a bottle, a vial, a syringe or test tube; and it may be amulti-use container. The pharmaceutical composition is preferablylyophilized.

Kits of the present invention preferably comprise a lyophilizedformulation of the present invention in a suitable container andinstructions for its reconstitution and/or use. Suitable containersinclude, for example, bottles, vials (e.g. dual chamber vials), syringes(such as dual chamber syringes) and test tubes. The container may beformed from a variety of materials such as glass or plastic. Preferablythe kit and/or container contain/s instructions on or associated withthe container that indicates directions for reconstitution and/or use.For example, the label may indicate that the lyophilized formulation isto be reconstituted to peptide concentrations as described above. Thelabel may further indicate that the formulation is useful or intendedfor subcutaneous administration.

The container holding the formulation may be a multi-use vial, whichallows for repeat administrations (e.g., from 2-6 administrations) ofthe reconstituted formulation. The kit may further comprise a secondcontainer comprising a suitable diluent (e.g., sodium bicarbonatesolution).

Upon mixing of the diluent and the lyophilized formulation, the finalpeptide concentration in the reconstituted formulation is preferably atleast 0.15 mg/mL/peptide (=75 μg) and preferably not more than 3mg/mL/peptide (=1500 μg). The kit may further include other materialsdesirable from a commercial and user standpoint, including otherbuffers, diluents, filters, needles, syringes, and package inserts withinstructions for use.

Kits of the present invention may have a single container that containsthe formulation of the pharmaceutical compositions according to thepresent invention with or without other components (e.g., othercompounds or pharmaceutical compositions of these other compounds) ormay have distinct container for each component.

Preferably, kits of the invention include a formulation of the inventionpackaged for use in combination with the co-administration of a secondcompound (such as adjuvants (e.g. GM-CSF), a chemotherapeutic agent, anatural product, a hormone or antagonist, an anti-angiogenesis agent orinhibitor, an apoptosis-inducing agent or a chelator) or apharmaceutical composition thereof. The components of the kit may bepre-complexed or each component may be in a separate distinct containerprior to administration to a patient. The components of the kit may beprovided in one or more liquid solutions, preferably, an aqueoussolution, more preferably, a sterile aqueous solution. The components ofthe kit may also be provided as solids, which may be converted intoliquids by addition of suitable solvents, which are preferably providedin another distinct container.

The container of a therapeutic kit may be a vial, test tube, flask,bottle, syringe, or any other means of enclosing a solid or liquid.Usually, when there is more than one component, the kit will contain asecond vial or other container, which allows for separate dosing. Thekit may also contain another container for a pharmaceutically acceptableliquid. Preferably, a therapeutic kit will contain an apparatus (e.g.,one or more needles, syringes, eye droppers, pipette, etc.), whichenables administration of the agents of the invention that arecomponents of the present kit.

The present formulation is one that is suitable for administration ofthe peptides by any acceptable route such as oral (enteral), nasal,ophthal, subcutaneous, intradermal, intramuscular, intravenous ortransdermal. Preferably, the administration is s.c., and most preferablyi.d. administration may be by infusion pump.

Since the peptides of the invention were isolated from lung cancer(including NSCLC and SCLC), the medicament of the invention ispreferably used to treat lung cancer (including NSCLC and SCLC).

The present invention further relates to a method for producing apersonalized pharmaceutical for an individual patient comprisingmanufacturing a pharmaceutical composition comprising at least onepeptide selected from a warehouse of pre-screened TUMAPs, wherein the atleast one peptide used in the pharmaceutical composition is selected forsuitability in the individual patient. In one embodiment, thepharmaceutical composition is a vaccine. The method could also beadapted to produce T cell clones for down-stream applications, such asTCR isolations, or soluble antibodies, and other treatment options.

A “personalized pharmaceutical” shall mean specifically tailoredtherapies for one individual patient that will only be used for therapyin such individual patient, including actively personalized cancervaccines and adoptive cellular therapies using autologous patienttissue.

As used herein, the term “warehouse” shall refer to a group or set ofpeptides that have been pre-screened for immunogenicity and/orover-presentation in a particular tumor type. The term “warehouse” isnot intended to imply that the particular peptides included in thevaccine have been pre-manufactured and stored in a physical facility,although that possibility is contemplated. It is expressly contemplatedthat the peptides may be manufactured de novo for each individualizedvaccine produced, or may be pre-manufactured and stored. The warehouse(e.g. in the form of a database) is composed of tumor-associatedpeptides which were highly overexpressed in the tumor tissue of lungcancer (including NSCLC and SCLC) patients with various HLA-A HLA-B andHLA-C alleles. It may contain MHC class I and MHC class II peptides orelongated MHC class I peptides. In addition to the tumor associatedpeptides collected from several lung cancers (including NSCLC and SCLC)tissues, the warehouse may contain HLA-A*02, HLA-A*01, HLA-A*03,HLA-A*24, HLA-B*07, HLA-B*08 and HLA-B*44 marker peptides. Thesepeptides allow comparison of the magnitude of T-cell immunity induced byTUMAPS in a quantitative manner and hence allow important conclusion tobe drawn on the capacity of the vaccine to elicit anti-tumor responses.Secondly, they function as important positive control peptides derivedfrom a “non-self” antigen in the case that any vaccine-induced T-cellresponses to TUMAPs derived from “self” antigens in a patient are notobserved. And thirdly, it may allow conclusions to be drawn, regardingthe status of immunocompetence of the patient.

TUMAPs for the warehouse are identified by using an integratedfunctional genomics approach combining gene expression analysis, massspectrometry, and T-cell immunology (XPresident®). The approach assuresthat only TUMAPs truly present on a high percentage of tumors but not oronly minimally expressed on normal tissue, are chosen for furtheranalysis. For initial peptide selection, lung cancer (including NSCLCand SCLC) samples from patients and blood from healthy donors wereanalyzed in a stepwise approach:

1. HLA ligands from the malignant material were identified by massspectrometry

2. Genome-wide messenger ribonucleic acid (mRNA) expression analysis wasused to identify genes over-expressed in the malignant tissue (lungcancer (including NSCLC and SCLC)) compared with a range of normalorgans and tissues

3. Identified HLA ligands were compared to gene expression data.Peptides over-presented or selectively presented on tumor tissue,preferably encoded by selectively expressed or over-expressed genes asdetected in step 2 were considered suitable TUMAP candidates for amulti-peptide vaccine.

4. Literature research was performed in order to identify additionalevidence supporting the relevance of the identified peptides as TUMAPs

5. The relevance of over-expression at the mRNA level was confirmed byredetection of selected TUMAPs from step 3 on tumor tissue and lack of(or infrequent) detection on healthy tissues.

6. In order to assess, whether an induction of in vivo T-cell responsesby the selected peptides may be feasible, in vitro immunogenicity assayswere performed using human T cells from healthy donors as well as fromlung cancer (including NSCLC and SCLC) patients.

In an aspect, the peptides are pre-screened for immunogenicity beforebeing included in the warehouse. By way of example, and not limitation,the immunogenicity of the peptides included in the warehouse isdetermined by a method comprising in vitro T-cell priming throughrepeated stimulations of CD8+ T cells from healthy donors withartificial antigen presenting cells loaded with peptide/MHC complexesand anti-CD28 antibody.

This method is preferred for rare cancers and patients with a rareexpression profile. In contrast to multi-peptide cocktails with a fixedcomposition as currently developed, the warehouse allows a significantlyhigher matching of the actual expression of antigens in the tumor withthe vaccine. Selected single or combinations of several “off-the-shelf”peptides will be used for each patient in a multitarget approach. Intheory, an approach based on selection of e.g. 5 different antigenicpeptides from a library of 50 would already lead to approximately 17million possible drug product (DP) compositions.

In an aspect, the peptides are selected for inclusion in the vaccinebased on their suitability for the individual patient based on themethod according to the present invention as described herein, or asbelow.

The HLA phenotype, transcriptomic and peptidomic data is gathered fromthe patient's tumor material, and blood samples to identify the mostsuitable peptides for each patient containing “warehouse” andpatient-unique (i.e. mutated) TUMAPs. Those peptides will be chosen,which are selectively or over-expressed in the patients' tumor and,where possible, show strong in vitro immunogenicity if tested with thepatients' individual PBMCs.

Preferably, the peptides included in the vaccine are identified by amethod comprising: (a) identifying tumor-associated peptides (TUMAPs)presented by a tumor sample from the individual patient; (b) comparingthe peptides identified in (a) with a warehouse (database) of peptidesas described above; and (c) selecting at least one peptide from thewarehouse (database) that correlates with a tumor-associated peptideidentified in the patient. For example, the TUMAPs presented by thetumor sample are identified by: (a1) comparing expression data from thetumor sample to expression data from a sample of normal tissuecorresponding to the tissue type of the tumor sample to identifyproteins that are over-expressed or aberrantly expressed in the tumorsample; and (a2) correlating the expression data with sequences of MHCligands bound to MHC class I and/or class II molecules in the tumorsample to identify MHC ligands derived from proteins over-expressed oraberrantly expressed by the tumor. Preferably, the sequences of MHCligands are identified by eluting bound peptides from MHC moleculesisolated from the tumor sample, and sequencing the eluted ligands.Preferably, the tumor sample and the normal tissue are obtained from thesame patient.

In addition to, or as an alternative to, selecting peptides using awarehousing (database) model, TUMAPs may be identified in the patient denovo, and then included in the vaccine. As one example, candidate TUMAPsmay be identified in the patient by (al) comparing expression data fromthe tumor sample to expression data from a sample of normal tissuecorresponding to the tissue type of the tumor sample to identifyproteins that are over-expressed or aberrantly expressed in the tumorsample; and (a2) correlating the expression data with sequences of MHCligands bound to MHC class I and/or class II molecules in the tumorsample to identify MHC ligands derived from proteins over-expressed oraberrantly expressed by the tumor. As another example, proteins may beidentified containing mutations that are unique to the tumor samplerelative to normal corresponding tissue from the individual patient, andTUMAPs can be identified that specifically target the mutation. Forexample, the genome of the tumor and of corresponding normal tissue canbe sequenced by whole genome sequencing: For discovery of non-synonymousmutations in the protein-coding regions of genes, genomic DNA and RNAare extracted from tumor tissues and normal non-mutated genomic germlineDNA is extracted from peripheral blood mononuclear cells (PBMCs). Theapplied NGS approach is confined to the re-sequencing of protein codingregions (exome re-sequencing). For this purpose, exonic DNA from humansamples is captured using vendor-supplied target enrichment kits,followed by sequencing with e.g. a HiSeq2000 (Illumina). Additionally,tumor mRNA is sequenced for direct quantification of gene expression andvalidation that mutated genes are expressed in the patients' tumors. Theresultant millions of sequence reads are processed through softwarealgorithms. The output list contains mutations and gene expression.Tumor-specific somatic mutations are determined by comparison with thePBMC-derived germline variations and prioritized. The de novo identifiedpeptides can then be tested for immunogenicity as described above forthe warehouse, and candidate TUMAPs possessing suitable immunogenicityare selected for inclusion in the vaccine.

In one exemplary embodiment, the peptides included in the vaccine areidentified by: (a) identifying tumor-associated peptides (TUMAPs)presented by a tumor sample from the individual patient by the method asdescribed above; (b) comparing the peptides identified in a) with awarehouse of peptides that have been prescreened for immunogenicity andoverpresentation in tumors as compared to corresponding normal tissue;(c) selecting at least one peptide from the warehouse that correlateswith a tumor-associated peptide identified in the patient; and (d)optionally, selecting at least one peptide identified de novo in (a)confirming its immunogenicity.

In one exemplary embodiment, the peptides included in the vaccine areidentified by: (a) identifying tumor-associated peptides (TUMAPs)presented by a tumor sample from the individual patient; and (b)selecting at least one peptide identified de novo in (a) and confirmingits immunogenicity.

Once the peptides for a personalized peptide based vaccine are selected,the vaccine is produced. The vaccine preferably is a liquid formulationconsisting of the individual peptides dissolved in between 20-40% DMSO,preferably about 30-35% DMSO, such as about 33% DMSO.

Each peptide to be included into a product is dissolved in DMSO. Theconcentration of the single peptide solutions has to be chosen dependingon the number of peptides to be included into the product. The singlepeptide-DMSO solutions are mixed in equal parts to achieve a solutioncontaining all peptides to be included in the product with aconcentration of ˜2.5 mg/ml per peptide. The mixed solution is thendiluted 1:3 with water for injection to achieve a concentration of 0.826mg/ml per peptide in 33% DMSO. The diluted solution is filtered througha 0.22 μm sterile filter. The final bulk solution is obtained.

Final bulk solution is filled into vials and stored at −20° C. untiluse. One vial contains 700 μL solution, containing 0.578 mg of eachpeptide. Of this, 500 μL (approx. 400 μg per peptide) will be appliedfor intradermal injection.

In addition to being useful for treating cancer, the peptides of thepresent invention are also useful as diagnostics. Since the peptideswere generated from lung cancer (including NSCLC and SCLC) cells andsince it was determined that these peptides are not or at lower levelspresent in normal tissues, these peptides can be used to diagnose thepresence of a cancer.

The presence of claimed peptides on tissue biopsies in blood samples canassist a pathologist in diagnosis of cancer. Detection of certainpeptides by means of antibodies, mass spectrometry or other methodsknown in the art can tell the pathologist that the tissue sample ismalignant or inflamed or generally diseased, or can be used as abiomarker for lung cancer (including NSCLC and SCLC). Presence of groupsof peptides can enable classification or sub-classification of diseasedtissues.

The detection of peptides on diseased tissue specimen can enable thedecision about the benefit of therapies involving the immune system,especially if T-lymphocytes are known or expected to be involved in themechanism of action. Loss of MHC expression is a well describedmechanism by which infected of malignant cells escapeimmuno-surveillance. Thus, presence of peptides shows that thismechanism is not exploited by the analyzed cells.

The peptides of the present invention might be used to analyzelymphocyte responses against those peptides such as T cell responses orantibody responses against the peptide or the peptide complexed to MHCmolecules. These lymphocyte responses can be used as prognostic markersfor decision on further therapy steps. These responses can also be usedas surrogate response markers in immunotherapy approaches aiming toinduce lymphocyte responses by different means, e.g. vaccination ofprotein, nucleic acids, autologous materials, adoptive transfer oflymphocytes. In gene therapy settings, lymphocyte responses againstpeptides can be considered in the assessment of side effects. Monitoringof lymphocyte responses might also be a valuable tool for follow-upexaminations of transplantation therapies, e.g. for the detection ofgraft versus host and host versus graft diseases.

The present invention will now be described in the following exampleswhich describe preferred embodiments thereof, and with reference to theaccompanying figures, nevertheless, without being limited thereto. Forthe purposes of the present invention, all references as cited hereinare incorporated by reference in their entireties.

FIGURES

FIGS. 1A through 1N show the over-presentation of various peptides indifferent cancer tissues (black dots). Upper part: Median MS signalintensities from technical replicate measurements are plotted as dotsfor single positive normal (grey dots) and tumor samples (black dots) onwhich the peptide was detected. Tumor and normal samples are groupedaccording to organ of origin, and box-and-whisker plots representmedian, 25th and 75th percentile (box), and minimum and maximum(whiskers) of normalized signal intensities over multiple samples.Normal organs are ordered according to risk categories (blood cells,blood vessels, brain, liver, lung: high risk, grey dots; reproductiveorgans, breast, prostate: low risk, grey dots; all other organs: mediumrisk; grey dots). Lower part: The relative peptide detection frequencyin every organ is shown as spine plot. Numbers below the panel indicatenumber of samples on which the peptide was detected out of the totalnumber of samples analyzed for each organ. If the peptide has beendetected on a sample but could not be quantified for technical reasons,the sample is included in this representation of detection frequency,but no dot is shown in the upper part of the figure. FIGS. 1A to 1B showthe over-presentation of various peptides in HLA-A*24 cancer tissuescompared to a panel of HLA-A*24 normal samples (N=19 for normal samples,N=94 for tumor samples). Tissues (from left to right): Normal samples:bloodvess (blood vessels); brain; heart; liver; lung; kidney; pituit(pituitary). Tumor samples: GBM: glioblastoma; GC: gastric cancer; HCC:hepatocellular carcinoma; NSCLCadeno (non-small cell lung canceradenocarcinoma); NSCLCother (non-small cell lung cancer); NSCLCsquam(non-small cell lung cancer squamous cell); SCLC: small cell lungcancer. FIG. 1A) Gene symbol: URB1, Peptide: LYQEILAQL (SEQ ID NO.: 62),FIG. 1B) Gene symbol: CKAP5, Peptide: VYPASKMFPFI (SEQ ID NO.: 65).FIGS. 1C to 1D show the over-presentation of various peptides inHLA-A*02 cancer tissues compared to a panel of HLA-A*02 normal samples(N=469 for normal samples, N=528 for tumor samples). Tissues (from leftto right): Normal samples: blood cells; bloodvess (blood vessels);brain; heart; liver; lung; adipose (adipose tissue); adren.gl. (adrenalgland); bile duct; bladder; BM (bone marrow); esoph (esophagus); eye;gallb (gallbladder); head&neck; kidney; large_int (large intestine); LN(lymph node); nerve; pancreas; parathyr (parathyroid gland); perit(peritoneum); pituit (pituitary); pleura; skel.mus (skeletal muscle);skin; small_int (small intestine); spleen; stomach; thyroid; trachea;ureter; breast; ovary; placenta; prostate; testis; thymus; uterus. Tumorsamples: AML: acute myeloid leukemia; BRCA: breast cancer; CCC:cholangiocellular carcinoma; CLL: chronic lymphocytic leukemia; CRC:colorectal cancer; GBC: gallbladder cancer; GBM: glioblastoma; GC:gastric cancer; GEJC: stomach cardia esophagus, cancer; HCC:hepatocellular carcinoma; HNSCC: head-and-neck cancer; MEL: melanoma;NHL: non-hodgkin lymphoma; NSCLCadeno (non-small cell lung canceradenocarcinoma); NSCLCother (non-small cell lung cancer); NSCLCsquam(non-small cell lung cancer squamous cell); OC: ovarian cancer; OSCAR:esophageal cancer; PACA: pancreatic cancer; PRCA: prostate cancer; RCC:renal cell carcinoma; SCLC: small cell lung cancer; UBC: urinary bladdercarcinoma; UEC: uterine and endometrial cancer. FIG. 1C) Gene symbol:BMS1, Peptide: VLYDKDAVYV (SEQ ID NO.: 129), FIG. 1D) Gene symbol:GORASP2, Peptide: NLWGGQGLLGV (SEQ ID NO.: 130). FIGS. 1E to 1F show theover-presentation of various peptides in HLA-A*01 cancer tissuescompared to a panel of HLA-A*01 normal samples (N=13 for normal samples,N=40 for tumor samples). Tissues (from left to right): Normal samples:blood cells; brain; heart; liver; lung. Tumor samples: GBM:glioblastoma; HCC: hepatocellular carcinoma; NSCLCadeno (non-small celllung cancer adenocarcinoma); NSCLCother (non-small cell lung cancer);NSCLCsquam (non-small cell lung cancer squamous cell); SCLC: small celllung cancer. FIG. 1E) Gene symbol: ZNF439, Peptide: LLDISQKNLY (SEQ IDNO.: 154), FIG. 1F) Gene symbol: MMP12, Peptide: SADDIRGIQSLY (SEQ IDNO.: 174). FIGS. 1G to 1H show the over-presentation of various peptidesin HLA-A*03 cancer tissues compared to a panel of HLA-A*03 normalsamples (N=12 for normal samples, N=28 for tumor samples). Tissues (fromleft to right): Normal samples: blood cells; bloodvess (blood vessels);brain; heart; liver; lung. Tumor samples: GBM: glioblastoma; GC: gastriccancer; NHL: non-hodgkin lymphoma; NSCLCadeno (non-small cell lungcancer adenocarcinoma); NSCLCother (non-small cell lung cancer);NSCLCsquam (non-small cell lung cancer squamous cell); SCLC: small celllung cancer. FIG. 1G) Gene symbols: KRT81, KRT121P, KRT83, KRT85, KRT86,Peptide: KLAELEGALQK (SEQ ID NO.: 202), FIG. 1H) Gene symbol: NDC80,Peptide: SINKPTSER (SEQ ID NO.: 457). FIGS. 1I to 1J show theover-presentation of various peptides in HLA-B*07 cancer tissuescompared to a panel of HLA-B*07 normal samples (N=13 for normal samples,N=36 for tumor samples). Tissues (from left to right): Normal samples:blood cells; bloodvess (blood vessels); brain; heart; liver; lung. Tumorsamples: GBM: glioblastoma; NSCLCadeno (non-small cell lung canceradenocarcinoma); NSCLCother (non-small cell lung cancer); NSCLCsquam(non-small cell lung cancer squamous cell); OC: ovarian cancer; SCLC:small cell lung cancer. FIG. 1I) Gene symbol: CTHRC1, Peptide: SPQRLRGLL(SEQ ID NO.: 291), FIG. 1J) Gene symbol: MANEA, Peptide: RPHKPGLYL (SEQID NO.: 475). FIGS. 1K to 1L show the over-presentation of variouspeptides in HLA-B*08 cancer tissues compared to a panel of HLA-B*08normal samples (N=1 for normal samples, N=22 for tumor samples). Tissues(from left to right): Normal samples: lung. Tumor samples: GBM:glioblastoma; NSCLCadeno (non-small cell lung cancer adenocarcinoma);NSCLCother (non-small cell lung cancer); NSCLCsquam (non-small cell lungcancer squamous cell); SCLC: small cell lung cancer. FIG. 1K) Genesymbol: VPS13B, Peptide: DIYQRALNL (SEQ ID NO.: 315), FIG. 1L) Genesymbol: ARID4A, Peptide: LVKVKVLL (SEQ ID NO.: 317). FIGS. 1M to 1N showthe over-presentation of various peptides in HLA-B*44 cancer tissuescompared to a panel of HLA-B*44 normal samples (N=15 for normal samples,N=25 for tumor samples). Tissues (from left to right): Normal samples:brain; heart; liver; lung. Tumor samples: GBM: glioblastoma; NSCLCadeno(non-small cell lung cancer adenocarcinoma); NSCLCother (non-small celllung cancer); NSCLCsquam (non-small cell lung cancer squamous cell);SCLC: small cell lung cancer. FIG. 1M) Gene symbol: NUP155, Peptide:SEKGVIQVY (SEQ ID NO.: 362), FIG. 1N) Gene symbol: CLSPN, Peptide:SEIGKAVGF (SEQ ID NO.: 489).

FIGS. 2A through 2N show exemplary exon expression profile of sourcegenes of the present invention that are over-expressed in differentcancer samples. Tumor (black dots) and normal (grey dots) samples aregrouped according to organ of origin, and box-and-whisker plotsrepresent median, 25th and 75th percentile (box), and minimum andmaximum (whiskers) RPKM values. Normal organs are ordered according torisk categories. FPKM=fragments per kilobase per million mapped reads.Normal samples: blood cells; bloodvess: blood vessel; brain; heart;liver; lung; adipose: adipose tissue; adren.gl.: adrenal gland; bileduct; bladder; BM: bone marrow; cartilage; esoph: esophagus; eye; gallb:gallbladder; head and neck; kidney; large_int: large intestine; LN:lymph node; nerve; pancreas; parathyr: parathyroid; perit: peritoneum;pituit: pituitary; pleura; skel.mus: skeletal muscle; skin; small_int:small intestine; spleen; stomach; thyroid; trachea; ureter; breast;ovary; placenta; prostate; testis; thymus; uterus. Tumor samples: AML:acute myeloid leukemia; BRCA: breast cancer; CCC: cholangiocellularcarcinoma; CLL: chronic lymphocytic leukemia; CRC: colorectal cancer;GBC: gallbladder cancer; GBM: glioblastoma; GC: gastric cancer; HCC:hepatocellular carcinoma; HNSCC: head-and-neck cancer; MEL: melanoma;NHL: non-hodgkin lymphoma; NSCLCadeno: non-small cell lung canceradenocarcinoma; NSCLCother: non-small cell lung cancer; NSCLCsquam:non-small cell lung cancer squamous cell; OC: ovarian cancer; OSCAR:esophageal cancer; PACA: pancreatic cancer; PRCA: prostate cancer; RCC:renal cell carcinoma; SCLC: small cell lung cancer; UBC: urinary bladdercarcinoma; UEC: uterine and endometrial cancer. FIG. 2A) Gene symbol:ADAMTS12, Peptide: QYDPTPLTW (SEQ ID No.: 1), FIG. 2B) Gene symbol:MMP12, Peptide: VWSNVTPLKF (SEQ ID No.: 2), FIG. 2C) Gene symbol: MMP12,Peptide: YVDINTFRL (SEQ ID No.: 84), FIG. 2D) Gene symbol: KIF26B,Peptide: TLYPYQISQL (SEQ ID No.: 87), FIG. 2E) Gene symbol: CT83,Peptide: NTDNNLAVY (SEQ ID No.: 164), FIG. 2F) Gene symbol: LAMA1,Peptide: VSDSECLSRY (SEQ ID No.: 189), FIG. 2G) Gene symbol: KIF26B,Peptide: KVKDTPGLGK (SEQ ID No.: 203), FIG. 2H) Gene symbol: SP6,Peptide: SLDGAARPK (SEQ ID No.: 205), FIG. 2I) Gene symbol: PRAME,Peptide: SPSVSQLSVL (SEQ ID No.: 220), FIG. 2J) Gene symbol: MMP1,Peptide: NPFYPEVEL (SEQ ID No.: 222), FIG. 2K) Gene symbol: NLRP2,Peptide: FNKRKPLSL (SEQ ID No.: 298), FIG. 2L) Gene symbol: KIF26B,Peptide: VASPKHCVL (SEQ ID No.: 300), FIG. 2M) Gene symbols: MAGEA3,MAGEA6, Peptide: MEVDPIGHVYIF (SEQ ID No.: 320), FIG. 2N) Gene symbol:MMP12, Peptide: QEMQHFLGL (SEQ ID No.: 326).

FIG. 3A and FIG. 3B show exemplary results of peptide-specific in vitroCD8+ T cell responses of a healthy HLA-A*02+ donor. CD8+ T cells wereprimed using artificial APCs coated with anti-CD28 mAb and HLA-A*02 incomplex with SeqID No 520 peptide (KIQEMQHFL, Seq ID NO: 520) (FIG. 3A,left panel). After three cycles of stimulation, the detection ofpeptide-reactive cells was performed by 2D multimer staining withA*02/SeqID 520 (FIG. 3A). Right panel (FIG. 3B) show control staining ofcells stimulated with irrelevant A*02/peptide complexes. Viable singletcells were gated for CD8+ lymphocytes. Boolean gates helped excludingfalse-positive events detected with multimers specific for differentpeptides. Frequencies of specific multimer+ cells among CD8+ lymphocytesare indicated.

FIG. 4A and FIG. 4B show exemplary results of peptide-specific in vitroCD8+ T cell responses of a healthy HLA-A*24+ donor. CD8+ T cells wereprimed using artificial APCs coated with anti-CD28 mAb and HLA-A*24 incomplex with SeqID No 504 peptide (FIG. 4A, left panel). After threecycles of stimulation, the detection of peptide-reactive cells wasperformed by 2D multimer staining with A*24/SeqID No 504 (VYEKNGYIYF,Seq ID NO: 504) (FIG. 4A). Right panel (FIG. 4B) shows control stainingof cells stimulated with irrelevant A*24/peptide complexes. Viablesinglet cells were gated for CD8+ lymphocytes. Boolean gates helpedexcluding false-positive events detected with multimers specific fordifferent peptides. Frequencies of specific multimer+ cells among CD8+lymphocytes are indicated.

FIG. 5A and FIG. 5B show exemplary results of peptide-specific in vitroCD8+ T cell responses of a healthy HLA-A*01+ donor. CD8+ T cells wereprimed using artificial APCs coated with anti-CD28 mAb and HLA-A*01 incomplex with SeqID No 153 peptide (KLDRSVFTAY, Seq ID NO: 153) (FIG. 5A,left panel) and SeqID No 173 peptide (RTEFNLNQY, Seq ID NO: 173) (FIG.5B, left panel), respectively. After three cycles of stimulation, thedetection of peptide-reactive cells was performed by 2D multimerstaining with A*01/SeqID No 153 (FIG. 5A) or A*01/SeqID No 173. Rightpanels (FIGS. 5A and 5B) show control staining of cells stimulated withirrelevant A*01/peptide complexes. Viable single cells were gated forCD8+ lymphocytes. Boolean gates helped excluding false-positive eventsdetected with multimers specific for different peptides. Frequencies ofspecific multimer+ cells among CD8+ lymphocytes are indicated.

FIG. 6A and FIG. 6B show exemplary results of peptide-specific in vitroCD8+ T cell responses of a healthy HLA-A*02+ donor. CD8+ T cells wereprimed using artificial APCs coated with anti-CD28 mAb and HLA-A*02 incomplex with SeqID No 89 peptide (ILSTTMVTV, Seq ID NO: 89) (FIG. 6A,left panel) and SeqID No 88 peptide (VQMVITEAQKV, Seq ID NO: 88) (FIG.6B, left panel), respectively. After three cycles of stimulation, thedetection of peptide-reactive cells was performed by 2D multimerstaining with A*02/SeqID No 89 (FIG. 6A) or A*02/SeqID No 88 (FIG. 6B).Right panels (FIGS. 6A and 6B) show control staining of cells stimulatedwith irrelevant A*02/peptide complexes. Viable single cells were gatedfor CD8+ lymphocytes. Boolean gates helped excluding false-positiveevents detected with multimers specific for different peptides.Frequencies of specific multimer+ cells among CD8+ lymphocytes areindicated.

FIG. 7A and FIG. 7B show exemplary results of peptide-specific in vitroCD8+ T cell responses of a healthy HLA-A*03+ donor. CD8+ T cells wereprimed using artificial APCs coated with anti-CD28 mAb and HLA-A*03 incomplex with SeqID No 208 peptide (GLASRILDAK, Seq ID NO: 208) (FIG. 7A,left panel) and SeqID No 210 peptide (ATSGVPVYK, Seq ID NO: 210) (FIG.7B, left panel), respectively. After three cycles of stimulation, thedetection of peptide-reactive cells was performed by 2D multimerstaining with A*03/SeqID No 208 (FIG. 7A) or A*03/SeqID No 210 (FIG.7B). Right panels (FIGS. 7A and 7B) show control staining of cellsstimulated with irrelevant A*03/peptide complexes. Viable single cellswere gated for CD8+ lymphocytes. Boolean gates helped excludingfalse-positive events detected with multimers specific for differentpeptides. Frequencies of specific multimer+ cells among CD8+ lymphocytesare indicated.

FIG. 8A and FIG. 8B show exemplary results of peptide-specific in vitroCD8+ T cell responses of a healthy HLA-A*24+ donor. CD8+ T cells wereprimed using artificial APCs coated with anti-CD28 mAb and HLA-A*24 incomplex with SeqID No 15 peptide (KYALLLQDL, Seq ID NO: 15) (FIG. 8A,left panel) and SeqID No 11 peptide (YYSKSVGFMQW, Seq ID NO: 11) (FIG.8B, left panel), respectively. After three cycles of stimulation, thedetection of peptide-reactive cells was performed by 2D multimerstaining with A*24/SeqID No 15 (FIG. 8A) or A*24/SeqID No 11 (FIG. 8B).Right panels (FIGS. 8A and 8B) show control staining of cells stimulatedwith irrelevant A*24/peptide complexes. Viable single cells were gatedfor CD8+ lymphocytes. Boolean gates helped excluding false-positiveevents detected with multimers specific for different peptides.Frequencies of specific multimer+ cells among CD8+ lymphocytes areindicated.

FIG. 9A and FIG. 9B show exemplary results of peptide-specific in vitroCD8+ T cell responses of a healthy HLA-B*07+ donor. CD8+ T cells wereprimed using artificial APCs coated with anti-CD28 mAb and HLA-B*07 incomplex with SeqID No 225 peptide (LPFDGPGGIL, Seq ID NO: 225) (FIG. 9A,left panel) and SeqID No 248 peptide (IPNWARQDL, Seq ID NO: 248) (FIG.9B, left panel), respectively. After three cycles of stimulation, thedetection of peptide-reactive cells was performed by 2D multimerstaining with B*07/SeqID No 225 (FIG. 9A) or B*07/SeqID No 248 (FIG.9B). Right panels (FIGS. 9A and 9B) show control staining of cellsstimulated with irrelevant B*07/peptide complexes. Viable single cellswere gated for CD8+ lymphocytes. Boolean gates helped excludingfalse-positive events detected with multimers specific for differentpeptides. Frequencies of specific multimer+ cells among CD8+ lymphocytesare indicated.

FIG. 10A and FIG. 10B show exemplary results of peptide-specific invitro CD8+ T cell responses of a healthy HLA-B*08+ donor. CD8+ T cellswere primed using artificial APCs coated with anti-CD28 mAb and HLA-B*08in complex with SeqID No 299 peptide (MAQFKEISL, Seq ID NO: 299) (FIG.10A, left panel) and SeqID No 297 peptide (RAQLKLVAL, Seq ID NO: 297)(FIG. 10B, left panel), respectively. After three cycles of stimulation,the detection of peptide-reactive cells was performed by 2D multimerstaining with B*08/SeqID No 299 (FIG. 10A) or B*08/SeqID No 297 (FIG.10B). Right panels (FIGS. 10A and 10B) show control staining of cellsstimulated with irrelevant B*08/peptide complexes. Viable single cellswere gated for CD8+ lymphocytes. Boolean gates helped excludingfalse-positive events detected with multimers specific for differentpeptides. Frequencies of specific multimer+ cells among CD8+ lymphocytesare indicated.

FIG. 11A and FIG. 11B show exemplary results of peptide-specific invitro CD8+ T cell responses of a healthy HLA-B*44+ donor. CD8+ T cellswere primed using artificial APCs coated with anti-CD28 mAb and HLA-B*44in complex with SeqID No 325 peptide (QEQDVDLVQKY, Seq ID NO: 325) (FIG.11A, left panel) and SeqID No 331 peptide (EDAQGHIW, Seq ID NO: 331)(FIG. 11B, left panel), respectively. After three cycles of stimulation,the detection of peptide-reactive cells was performed by 2D multimerstaining single cells were gated for CD8+ lymphocyteswith B*44/SeqID No325 (FIG. 11A) or B*44/SeqID No 331 (FIG. 11B). Right panels (FIGS. 11Aand 11B) show control staining of cells stimulated with irrelevantB*44/peptide complexes. Viable single cells were gated for CD8+lymphocytes. Boolean gates helped excluding false-positive eventsdetected with multimers specific for different peptides. Frequencies ofspecific multimer+ cells among CD8+ lymphocytes are indicated.

EXAMPLES Example 1

Identification and Quantitation of Tumor Associated Peptides Presentedon the Cell Surface

Tissue Samples

Patients' tumor tissues were obtained from: Asterand (Detroit, Mich.,USA & Royston, Herts, UK); Bio-Options Inc. (Brea, Calif., USA);Geneticist Inc. (Glendale, Calif., USA); University Hospital Heidelberg(Heidelberg, Germany); ProteoGenex Inc. (Culver City, Calif., USA);Tissue Solutions Ltd (Glasgow, UK); University Hospital Munich (Munich,Germany). Normal tissues were obtained from Asterand (Detroit, Mich.,USA & Royston, Herts, UK); Bio-Options Inc. (Brea, Calif., USA);BioServe (Beltsville, Md., USA); Capital BioScience Inc. (Rockville,Md., USA); Centre for Clinical Transfusion Medicine Tuebingen (Tübingen,Germany); Geneticist Inc. (Glendale, Calif., USA); Kyoto PrefecturalUniversity of Medicine (KPUM) (Kyoto, Japan); Osaka City University(OCU) (Osaka, Japan); ProteoGenex Inc. (Culver City, Calif., USA);Tissue Solutions Ltd (Glasgow, UK); University Hospital Geneva (Geneva,Switzerland); University Hospital Heidelberg (Heidelberg, Germany);University Hospital Tübingen (Tubingen, Germany); University HospitalMunich (Munich, Germany). Written informed consents of all patients hadbeen given before surgery or autopsy. Tissues were shock-frozenimmediately after excision and stored until isolation of TUMAPs at −70°C. or below.

Isolation of HLA Peptides from Tissue Samples

HLA peptide pools from shock-frozen tissue samples were obtained byimmune precipitation from solid tissues according to a slightly modifiedprotocol (Falk et al., 1991; Seeger et al., 1999) using theHLA-A*02-specific antibody BB7.2, the HLA-A, —B, C-specific antibodyW6/32, the HLA-DR specific antibody L243 and the HLA DP specificantibody B7/21, CNBr-activated sepharose, acid treatment, andultrafiltration.

Mass Spectrometry Analyses

The HLA peptide pools as obtained were separated according to theirhydrophobicity by reversed-phase chromatography (nanoAcquity UPLCsystem, Waters) and the eluting peptides were analyzed in LTQ-velos andfusion hybrid mass spectrometers (ThermoElectron) equipped with an ESIsource. Peptide pools were loaded directly onto the analyticalfused-silica micro-capillary column (75 m i.d.×250 mm) packed with 1.7μm C18 reversed-phase material (Waters) applying a flow rate of 400 nLper minute. Subsequently, the peptides were separated using a two-step180 minute-binary gradient from 10% to 33% B at a flow rate of 300 nLper minute. The gradient was composed of Solvent A (0.1% formic acid inwater) and solvent B (0.1% formic acid in acetonitrile). A gold coatedglass capillary (PicoTip, New Objective) was used for introduction intothe nanoESI source. The LTQ-Orbitrap mass spectrometers were operated inthe data-dependent mode using a TOP5 strategy. In brief, a scan cyclewas initiated with a full scan of high mass accuracy in the orbitrap(R=30 000), which was followed by MS/MS scans also in the orbitrap(R=7500) on the 5 most abundant precursor ions with dynamic exclusion ofpreviously selected ions. Tandem mass spectra were interpreted bySEQUEST at a fixed false discovery rate (q≤0.05) and additional manualcontrol. In cases where the identified peptide sequence was uncertain itwas additionally validated by comparison of the generated naturalpeptide fragmentation pattern with the fragmentation pattern of asynthetic sequence-identical reference peptide.

Label-free relative LC-MS quantitation was performed by ion countingi.e. by extraction and analysis of LC-MS features (Mueller et al.,2007). The method assumes that the peptide's LC-MS signal areacorrelates with its abundance in the sample. Extracted features werefurther processed by charge state deconvolution and retention timealignment (Mueller et al., 2008; Sturm et al., 2008). Finally, all LC-MSfeatures were cross-referenced with the sequence identification resultsto combine quantitative data of different samples and tissues to peptidepresentation profiles. The quantitative data were normalized in atwo-tier fashion according to central tendency to account for variationwithin technical and biological replicates. Thus, each identifiedpeptide can be associated with quantitative data allowing relativequantification between samples and tissues. In addition, allquantitative data acquired for peptide candidates was inspected manuallyto assure data consistency and to verify the accuracy of the automatedanalysis. For each peptide, a presentation profile was calculatedshowing the mean sample presentation as well as replicate variations.The profiles juxtapose cancer samples to a baseline of normal tissuesamples. Presentation profiles of peptides exemplary over-presented orexclusively presented on tumors are shown in FIGS. 1A through 1N.

Table 8 shows the presentation on various cancer entities for selectedpeptides, and thus the particular relevance of the peptides as mentionedfor the diagnosis and/or treatment of the cancers as indicated (e.g.peptide SEQ ID No. 3 for hepatocellular carcinoma, peptide SEQ ID No. 11for melanoma, ovarian cancer and uterine cancer).

TABLE 8Overview of presentation of selected tumor-associated peptides of the presentinvention across entities. AML = acute myeloid leukemia, BRCA =breast cancer, CCC = bile duct cancer, GBM = brain cancer, CLL =chronic lymphocytic leukemia, CRC = colorectal carcinoma, OSCAR =esophageal cancer, GBC = gallbladder adenocarcinoma, GC =gastric cancer, HNSCC = head and neck squamous cell carcinoma, HCC =hepatocellular carcinoma, MEL = melanoma, NHL = non-Hodgkinlymphoma, OC = ovarian cancer, PACA = pancreatic cancer, PRCA = prostatecancer and benign prostate hyperplasia, RCC =renal cell carcinoma, UBC = urinary bladder cancer, UEC = uterinecancer.SEQ ID No. Sequence Peptide Presentation on cancer entities   3 YLEKFYGLHCC   6 KYKDYFPVI HCC   9 RILRFPWQL MEL  11 YYSKSVGFMQW MEL, OC, UEC  13HYTYILEVF GC, UEC  14 SYSSCYSF GC  18 DYIGSVEKW PRCA  19 ILKEDPFLFOC, RCC  21 SYEVRSTF CCC, OC, PRCA  22 TQPGDWTLF MEL  23 KFIISDWRF MEL 24 MYPDLSELLM AML, GBM, OC, UEC  26 KTPTNYYLF GC  28 YYSIISHTL HCC  31QYQNVLTLW GBM, GC, HCC, MEL, NHL, PRCA, RCC, UEC  32 SLPDLTPTF PRCA  33KSSVIASLLF GBM  34 MQPRMFFLF AML, GBM, HCC, MEL, UEC  36 KQMEDGHTLFAML, OC  37 QWPWQASLQF GC  38 KYTNWKAFL AML, HCC  41 VIYFMGAIFHCC, PRCA, UEC  43 IQMDEPMAF AML, MEL, OC  44 AYLSAVGTF AML, GC, MEL  45KYFVPPQLF GC  47 KYADYFLEV GBM, OC, UEC  48 VFIDHPVHLKF UEC  50SYPELVKMVW AML, GC, HCC  51 KYALLLQELAML, CLL, GBM, GC, HCC, MEL, OC, PRCA, RCC, UEC  52 KYMKIFHKF OC, UEC 53 KYITNLEDL PRCA  54 LLIKLLQTF AML, GBM, MEL, OC, PRCA, RCC  55RWMDQRLVF GC, HCC, MEL, UEC  56 VYMIEPLEL GBM, NHL  57 YPSIIQEF GBM, RCC 58 QFAAPLRGIYF GBM, HCC  59 KYSTTFFMV GBM  60 TYLSIFDQLAML, GC, HCC, OC  61 NYAENILTL AML, GBM, GC, MEL  62 LYQEILAQLAML, GBM, GC, HCC, MEL, PRCA, RCC  63 VMPSDSFFF MEL, NHL, OC, UEC  64NYAIFDEGHML GBM, GC  65 VYPASKMFPFI GBM, GC, HCC, MEL, NHL, OC, UEC  66IYFRDSSFL AML, GC, MEL  67 RYPGKFYRV OC  68 IYQQIIQTY GC, MEL, UEC  69IMPEKFEFW AML, GC, MEL, NHL, UEC  70 PYTNYTFDF GC, MEL  71 SYMVLAPVF MEL 72 RYEGILYTI GC, HCC, NHL, PRCA  73 SYIGLPLTL GC, HCC, RCC  74VYDQYFITL AML, PRCA  76 WYGWHFPEL AML, GC, HCC, RCC, UEC  77 AYTLLGHEFVGC, MEL, OC  78 TWFPKTPMLF AML, GBM, GC, HCC, MEL, UEC  79 RYLADLPTLGC, HCC, OC, UEC  80 YYSPLRDLL MEL  82 RFLPSPVVIAML, GC, HCC, MEL, PRCA, UEC  83 TYCQNIKEF AML, OC, PRCA, UEC  84YVDINTFRL HCC  86 FVIDGFDEL CRC, GC, NHL, OSCAR  87 TLYPYQISQLHCC, OSCAR  90 FLLMHPSI CLL, HCC, RCC  91 FALPGLLHAGC, HCC, NHL, OSCAR, RCC  92 NLRDLLSEV HCC  93 TLQEKILQV CLL, GBM, NHL 95 ITIGVLARV CRC, PACA  96 HLVGGLHTV AML, BRCA, CRC, GC, MEL, OC, PRCA 97 VLALVNSTV CLL  98 LQSSGLTLLL GBM, OC, PRCA  99 FLKEKVPGICLL, GC, MEL, NHL 100 RQYPTPFQL AML, CLL, CRC, GBM, NHL, OC, RCC 101FIISDWRFVL HNSCC 102 SLLEQAIAL GC, HCC, NHL, OC, UEC 103 FLYYPDPVLGBC, HCC, MEL, NHL 105 SLLTHIPTAAML, CRC, HNSCC, MEL, OC, OSCAR, RCC, UBC, UEC 106 FIIDTTYPAYVCRC, OC, OSCAR 107 LLQGAIESV BRCA, CLL, CRC, HNSCC, MEL, NHL, UBC, UEC108 MIIALSLYI AML, BRCA 110 LLADFQALL RCC 111 ALCLLLHLLAML, GBC, HCC, HNSCC, RCC 113 AVLTGLVEVBRCA, CLL, CRC, GC, HCC, NHL, OSCAR, RCC, UEC 114 ILDERQVLLAML, BRCA, CRC, GBC, GC, HCC, HNSCC, MEL, NHL, OC, OSCAR, PACA, UBC, UEC115 MLLETQDALYV HNSCC 116 VLMEENSKL GBM 117 FLDPNARPLV NHL, OC 118ALSSVLHSI AML, BRCA, CRC, GBC, HCC, HNSCC, MEL, NHL, OC, OSCAR,PACA, RCC, UBC 119 RTADITVTV AML, CRC, UBC 120 ALLANLPAVGBC, GC, OC, PACA 121 ALVDTLTGI HNSCC, NHL, UEC 122 ALLEMFPEITVBRCA, OC, PRCA 123 LMAFFLAVV CCC, HCC, NHL, OSCAR, RCC 124 SVASVLLYLAML, BRCA, CLL, HCC, HNSCC, NHL, OC 125 VLQPFLPSIAML, BRCA, CCC, CLL, CRC, HCC, HNSCC, MEL, NHL, OC, RCC 126 FLSTVTSVCCC, GBM, HCC, HNSCC, MEL, NHL, OSCAR, PACA, RCC, UBC, UEC 127 GLDGSLVFLAML, CLL, CRC, GBM, HCC, HNSCC, MEL, NHL, OC, OSCAR, UBC 128 FLGTTPTLAML, CLL, GBM, HNSCC, NHL, OC, UBC, UEC 129 VLYDKDAVYVAML, CLL, CRC, HNSCC, NHL, OC, UBC, UEC 130 NLWGGQGLLGVBRCA, GBC, GBM, GC, HCC, OC, PRCA, UEC 131 LLKEFVQRVBRCA, CRC, HCC, HNSCC, OC, OSCAR 132 ALWLVDPLTV CLL, CRC, GBM, HCC 133MTLPVDAVISV CLL, CRC, HCC, HNSCC, NHL, PRCA, UEC 134 AAEIGDKSWLYGC, MEL, NHL, UEC 135 ASEDSVLLY GBM, GC, MEL, NHL, OSCAR, PRCA, 136ATDLVVLDRY GBM, GC, HCC, MEL, NHL, OSCAR, PRCA, UEC 137 ATSKFMEFYGBM, MEL, OC, PRCA, UEC 139 ECDMAFHIY MEL, OC 140 ESDREELNY GC, PRCA 141ESDVGVVVY GBM, GC 142 EVAEPSVLFDLY GC, MEL, NHL, OC, UEC 144 FLDSQNLSAYGC 145 FVDKPVAY GBM, GC, MEL 146 GLNTGSALSY GC, OC 148 GTEFTTILYGC, NHL, OSCAR, PRCA 149 GTEFTTVLY GC, HNSCC, MEL, OSCAR, PRCA 150GTELLSLVY GC, MEL, NHL, OC, PRCA, UEC 152 HTDSLHLLI GC, MEL, OC 154LLDISQKNLY GBM, NHL, PRCA 155 LLDPNPHMY PRCA 156 LLDSLREQYGC, NHL, OSCAR 157 LMDRPIFY GBM, GC, MEL, NHL 159 LSDTSVIQFYGBM, GC, HCC, MEL, NHL, PRCA 160 LTEAVLNRY OC 161 LVDDGTHGQYGBM, GC, MEL 162 LVDNSIRELQY GC, HCC, MEL, NHL, OC, UEC 163 NSDSSLTLREFYHCC, PRCA 166 NTQITDIGRY MEL 167 QSDPGTSVLGY GBM 169 RLDTPLYFSY MEL, OC170 RSDDTAVYY CLL, GC, HCC, NHL, PRCA, UEC 172 RTDSCSSAQAQY MEL 173RTEFNLNQY GBC, GC, MEL, UEC 177 SSDEVNFLVY GC, MEL, OC, UEC 178SSDSSTLPKL GC, OC, PRCA 179 STAKSATWTY PRCA 180 STDPWIQMAY GBM, GC, MEL181 TADGKTYYY GBM, HCC, MEL, PRCA 182 TDYHVRVY GBM, PRCA 184 TSAHPEDSSFYGC, NHL, PRCA 186 TTDIIEKY GC, MEL, 187 VADLHLYLY GC, MEL, OC, PRCA, RCC190 VTDGINPLIDRY MEL 191 VTDGSLYEGVAY GC, MEL, UEC 192 VTEESFDSKFY GC193 VTEFSLNTY GBC, GC, MEL, OC, OSCAR, UEC 196 WMFFVINY UEC 197YADTVRPEFY OC 198 YLDPVQRDLY GBM, GC, HCC, MEL, NHL, OC, UEC 202KLAELEGALQK MEL, OC, UEC 204 AVFDKFIRY GBM 207 RSFNGLLTMY MEL, OC 210ATSGVPVYK UEC 211 TVNPVAIHK GBM, NHL, OC, PRCA, UEC 212 KAYEQVMHY UEC213 LNINMTSPMGTK GBM, GC, MEL, NHL, PACA 214 RTMSEAALVRKGC, NHL, OC, PRCA, UEC 215 MMFSGPQILKL MEL 216 KLYAWELAFAML, GBM, MEL, OC, PRCA 217 RILNQILYYAML, GBM, GC, HCC, MEL, NHL, PRCA, UEC 218 KTLVAELLILK AML, NHL, UEC 219RLRSSLVFK UEC 220 SPSVSQLSVL UEC 235 MPLKHYLLL MEL, NHL, OC, UEC 237RPAATAVISL GBM, NHL, OC 244 FPYVRDFVM GBC, MEL, NHL, OC, UEC 247RALLARLLL NHL 251 VPRSSGQTV BRCA, GBM, UEC 255 MPLLENLYL OC, UEC 256SPRVPSIEL NHL 259 RPPAAGLRGISL GBM 260 YPQHPGLNA BRCA, GBM, GC, NHL 262SAYPQRLEI GC 263 HPAPYGDLL UEC 271 MPLPWSLALP MEL 273 MPLLWLRGF UEC 274TPYQEHVAL OC 275 APHPPLSVV AML, BRCA, MEL 276 LPRAGGAFLNHL, OC, RCC, UEC 277 MPLFEPRVF OC, UEC 278 HPMIDINGIIVF UEC 280VPISEEGTPVL MEL, OC, PRCA, UEC 281 RPRAPVTPA GBM 282 MPQIETRVIL UEC 283RPHSLSSEL AML, NHL 284 FPVTSIFHTF MEL, OC, UEC 285 FPSFLTNSL AML 286VPTLRSEL OC 288 FPQKFIDLL UEC 289 VPENHSVAL UEC 290 APYRPPDISL BRCA 292SPQRLRGLLL NHL 293 RPRSALPRLLLP NHL, UEC 295 KPEGTRIAV NHL, UEC 296MPMQDIKM UEC 300 VASPKHCVL OC 301 YMHKLLVL AML, NHL, OC, PRCA, UEC 305ALKLRVAVL NHL 306 ILKVKVGL MEL, OC 308 MLKQKVEEL OSCAR 311 EIRIRVVQMMEL, NHL, OC, PRCA 313 ELKKKEYEEL NHL 314 AIISRLVAL NHL, OSCAR, UBC 316VIKEKALTL NHL, OC, PRCA, RCC 318 EAAIRSVEL GBM, MEL, NHL, OC 321AEMLESVIKNY NHL 322 KEVDPAGHSY NHL 323 SEFMQVIF NHL 328 FEYDFLLQRI UEC330 KEGDLGGKQW NHL 335 KELEATKQY MEL, NHL 337 TENRYCVQL HCC 342HEFSSPSHL NHL 343 TEFTTVLY GBM, NHL, PRCA 345 IEFIHPQAFGBM, GC, NHL, PRCA 347 ALNPYQYQY UEC 348 AEIQGNINHV UEC 351 EEVNYINTFAML, MEL, NHL, OC 354 TEDPTILRI GC, HCC, OC, PRCA, UEC 356 EEGRVYLFGBM, PRCA 357 RELENCFQIQ UEC 359 DELFSIALY NHL 363 AELDKLTSV GBM, UEC366 AENLFRAF GBM, NHL, OC 367 GEVHPSEMI UEC 368 GEFPVRVQVAML, GC, OC, UEC 370 YEDLSQKY GBM, NHL, OC 371 GELALKKKI UEC 372TEGIIMKDF OC, PRCA, RCC, UEC 373 MEMQKSPVF NHL, OC 374 DEVNFLVYCCC, GBC, GBM, NHL, OC, PRCA 375 VYSDLHAFYY GBM, GC, HCC, MEL, PRCA 376KYVKDFHKF AML, OC, PRCA, UEC 377 VYVGAVNRI GC, HCC, PRCA 378 KFLGPAEHLTFOC 379 NYIVPDKQIF GBM, GC, HCC, MEL, OC, PRCA 380 VFQEKHHVI PRCA 381TYSKKHFRI MEL, OC 382 IYHSHHPTL AML, MEL, NHL, OC, UEC 383 RYKQDVERFAML, MEL, OC, PRCA, UEC 384 KYVKVFDKF AML, UEC 385 MYINEVERLGBM, MEL, OC, PRCA, UEC 386 VYNDHSIYVWAML, GBM, HCC, MEL, NHL, PRCA, UEC 387 RWLPQKNAAQFAML, GC, HCC, MEL, OC, RCC, UEC 388 FSIPEGALVAVAML, CCC, CLL, CRC, GBC, GC, HCC, HNSCC, MEL, NHL, OC,OSCAR, PRCA, RCC, UBC, UEC 389 TLMEQPLTTLAML, BRCA, CRC, HCC, HNSCC, NHL, OC, PRCA, UEC 390 HIMPTVHTVBRCA, CLL, GBM, HCC, HNSCC, MEL, NHL, OC, OSCAR, UBC, UEC 391SLIDMRGIETV BRCA, CLL, GBM, HCC, HNSCC, OC, UBC 392 SLFKDQMELAML, BRCA, CLL, CRC, GBM, HCC, MEL, NHL, OC, PRCA, RCC, UBC, UEC 393ILLPYLQTL AML, BRCA, CLL, CRC, GBM, HCC, HNSCC, NHL, OC 394 ASEAEMRLFYGBM, GC, MEL, NHL 395 ASEASRLAHY GBM, GC, NHL, PRCA, UEC 396 ASEFGNHYLYGBM, GC, MEL, UEC 397 ASEITSKGASLY GBM, GC, HCC, MEL, OC, PRCA 398ASEQQALHTVQY GBM, GC, MEL, NHL, PRCA, UEC 399 ATDIPCLLY MEL 400ATDISRQNEY GBM, GC, NHL, OC 401 DSDESYMEKSLY GC 402 DTDSQRLAYGBM, GC, MEL 403 ELDSKVEVLTY GBM, GC, MEL, OC, PRCA 404 ETARKFLYY GBM405 ETEEGIYWRY GC 406 ETEQTKFWDY GBM, GC, MEL, OC, RCC, UEC 407FSDNDKLYLY GBM, GC, MEL, NHL, PRCA, UEC 408 FTEQWTDGYGBM, GC, OC, OSCAR, PRCA, 409 FVDPLVTNYGBM, GC, HCC, MEL, NHL, OC, OSCAR, PRCA, RCC 410 GSDHQSPSSSSYGBM, GC, MEL, OC, PRCA, 411 GTVYEDLRY GBM, GC, HCC, MEL, NHL, OC, OSCAR412 ILDEVIMGY GBM, GC, MEL, NHL, OC, OSCAR 413 ISDRYYTALYGBM, GC, PRCA, UEC 414 KTDESLTKY GC, NHL, PRCA 415 LLDPRSYHTY GC, NHL416 LLDTAQKNLY GBM, NHL, PRCA 417 LLEDKHFQSYGBM, GC, HCC, MEL, NHL, OC, PRCA, UEC 418 LSDPSGPKSY GBM, HCC, PRCA 419LSELKPMSY GBM, GC, HCC, MEL, OSCAR, PRCA, RCC, UEC 420 LTEDKETLQYGC, HCC, MEL, NHL, PRCA 421 LTELLERAAFY GC, MEL, NHL, UEC 422 MIDVTKSYYGBM, GC, MEL, NHL, OC, OSCAR, PRCA, UEC 423 NLDAVHDITVAYGC, MEL, PRCA, UEC 424 NLDEEKQLLY MEL, PRCA 425 NLDIIQQEYGBM, GC, HCC, MEL, NHL, OC, OSCAR, PRCA, UEC 426 NLDQATRVAY NHL 427NSDEQKITEMVY GC 428 NSELSCQLY GBM, GC, MEL, RCC 429 NTEDSSMSGYLYGC, MEL, 430 NTEGLHHLY GBM, HCC, MEL, PRCA 431 NTSDMMGRMSYGC, MEL, NHL, OC, 432 NVDPVQHTYGBM, GC, HCC, HNSCC, MEL, NHL, OC, OSCAR, PRCA, RCC, UEC 433 QIDTGENLYGC, MEL 434 QTDCAPNNGY GBM, GC, MEL, PRCA 435 QTDDTWRTEYGBM, GC, MEL, NHL, PRCA 436 QTETGTPYMLYGBM, GC, MEL, NHL, OC, OSCAR, PRCA, UEC 437 STDGKHVWVEYGC, MEL, NHL, PRCA 438 STDNFNCKY GC, HCC, MEL 439 TLDAGKFQIYGC, HCC, MEL, NHL, PRCA, UEC 440 TLDENPGVRYGBM, GC, HCC, MEL, NHL, PRCA, UEC 441 TLDSALNAASYYGBM, GC, MEL, NHL, PRCA, RCC 442 TSDFSRFTNY GBM 443 TTDFPSESSFEYGC, MEL, NHL, OC, PRCA, UEC 444 TTDTVIRSY GC, MEL, UEC 445 VLDQGKITEYGBM, HCC 446 VTAQVVGTERY GC, MEL 447 VVDEDHELIY GBM 448 YLDIPNPRYGBM, MEL, RCC 449 YLDRGTGNVSFY GBM, GC, HCC, MEL, PRCA, RCC 450YSDDGQKWTVY MEL 451 YSDSLVQKGY GBM, GC, HCC, OC, PRCA, UEC 452YVDAVLGKGHQY GBM, GC, MEL, NHL, OC, PRCA, UEC 453 AINTSIKNK PRCA 454KVYTPSISK GBM, HCC, MEL, UEC 455 RIADIFVKK GC, MEL, NHL, OC, UEC 456SMFTAILKK AML, MEL, NHL, OC, UEC 457 SINKPTSER GBM 458 GIADFVLKYAML, BRCA, GBM, MEL, NHL, UEC 461 RPILIIVTL NHL 464 YPRPGTPAAAML, GC, MEL, NHL, OC, RCC 465 VPRPIFSQL NHL 468 SPMYGQAGL RCC 469YPENGVVQM AML, OC 470 SPNSYFRVL RCC 471 KPRPDVTNEL NHL 472 NPRATDAQL AML473 LPRALLSSL NHL, OC 474 LPRLLPAL AML, NHL 476 AEEEIMKKI NHL 477QENSYQSRL OC 479 AEIQPQTQV UEC 480 GEVSGLTKDF MEL, NHL, OC 481 RELQHEHSLOC 482 TEREWADEW AML, MEL, NHL, OC, RCC 483 EENDQSTHKWMEL, NHL, OC, RCC, UEC 484 AEVGFVRFF AML, MEL, NHL, OC 485 SEIEDSTKQVFMEL, NHL, OC 486 SEDDPILQI NHL, OC, UEC 487 AEDQLHHSF AML, NHL 488TEFPIIKMY AML, MEL, NHL, OC, PRCA

Example 2

Expression Profiling of Genes Encoding the Peptides of the Invention

Over-presentation or specific presentation of a peptide on tumor cellscompared to normal cells is sufficient for its usefulness inimmunotherapy, and some peptides are tumor-specific despite their sourceprotein occurring also in normal tissues. Still, mRNA expressionprofiling adds an additional level of safety in selection of peptidetargets for immunotherapies. Especially for therapeutic options withhigh safety risks, such as affinity-matured TCRs, the ideal targetpeptide will be derived from a protein that is unique to the tumor andnot found on normal tissues.

RNA Sources and Preparation

Surgically removed tissue specimens were provided as indicated above(see Example 1) after written informed consent had been obtained fromeach patient. Tumor tissue specimens were snap-frozen immediately aftersurgery and later homogenized with mortar and pestle under liquidnitrogen. Total RNA was prepared from these samples using TRI Reagent(Ambion, Darmstadt, Germany) followed by a cleanup with RNeasy (QIAGEN,Hilden, Germany); both methods were performed according to themanufacturer's protocol.

Total RNA from healthy human tissues for RNASeq experiments was obtainedfrom: Asterand (Detroit, Mich., USA & Royston, Herts, UK); Bio-OptionsInc. (Brea, Calif., USA); BioCat GmbH (Heidelberg, Germany); BioServe(Beltsville, Md., USA); Capital BioScience Inc. (Rockville, Md., USA);Geneticist Inc. (Glendale, Calif., USA); Heidelberg University Hospital(Thoraxklinik, Heidelberg, Germany); Istituto Nazionale Tumori “Pascale”(Naples, Italy); ProteoGenex Inc. (Culver City, Calif., USA).

Total RNA from tumor tissues for RNASeq experiments was obtained from:Asterand (Detroit, Mich., USA & Royston, Herts, UK); Geneticist Inc.(Glendale, Calif., USA); ProteoGenex Inc. (Culver City, Calif., USA);Tissue Solutions Ltd (Glasgow, UK); University Hospital Bonn (Bonn,Germany); University Hospital Thbingen (Thbingen, Germany).

Quality and quantity of all RNA samples were assessed on an Agilent 2100Bioanalyzer (Agilent, Waldbronn, Germany) using the RNA 6000 PicoLabChip Kit (Agilent).

RNAseq Experiments

Gene expression analysis of—tumor and normal tissue RNA samples wasperformed by next generation sequencing (RNAseq) by CeGaT (Thbingen,Germany). Briefly, sequencing libraries are prepared using the IlluminaHiSeq v4 reagent kit according to the provider's protocol (IlluminaInc., San Diego, Calif., USA), which includes RNA fragmentation, cDNAconversion and addition of sequencing adaptors. Libraries derived frommultiple samples are mixed equimolar and sequenced on the Illumina HiSeq2500 sequencer according to the manufacturer's instructions, generating50 bp single end reads. Processed reads are mapped to the human genome(GRCh38) using the STAR software. Expression data are provided ontranscript level as RPKM (Reads Per Kilobase per Million mapped reads,generated by the software Cufflinks) and on exon level (total reads,generated by the software Bedtools), based on annotations of the ensemblsequence database (Ensembl77). Exon reads are normalized for exon lengthand alignment size to obtain RPKM values.

Exemplary expression profiles of source genes of the present inventionthat are highly over-expressed or exclusively expressed in lung cancer(including NSCLC and SCLC) are shown in FIGS. 2A through 2N. Expressionscores for further exemplary genes are shown in Table 9.

TABLE 9Expression scores. The table lists peptides from genes that are veryhighly over-expressed in lung cancer tissues (NSCLCadeno = non-smallcell lung carcinoma adenocarcinoma; NSCLCsquam = non-small celllung carcinoma squamous cell; NSCLCother = non-small cell lungcarcinoma, other subtypes; SCLC = small cell lung carcinoma)compared to a panel of normal tissues (+++), highly over-expressedin tumors compared to a panel of normal tissues (++) or over-expressedin tumors compared to a panel of normal tissues (+). The baselinefor this score was calculated from measurements of the followingrelevant normal tissues: adipose tissue, adrenal gland, bile duct,blood cells, blood vessels, bone marrow, brain, cartilage, esophagus,eye, gallbladder, heart, head&neck, kidney, large intestine, liver,lung, lymph node, nerve, parathyroid, pancreas, pituitary, pleura,skeletal muscle, skin, small intestine, spleen, stomach, thyroid gland,trachea, urinary bladder, ureter. In case expression data for severalsamples of the same tissue type were available, the arithmetic mean ofall respective samples was used for the calculation. Gene ExpressionSEQ ID No Sequence NSCLCadeno NSCLCsquam NSCLCother SCLC   1 QYDPTPLTW+++ +++ + +   2 VWSNVTPLKF +++ +++ + +++   3 YLEKFYGL +++ +++ +++   4SYEKVINYL +++ +++   5 RYMKKDYLI +++   6 KYKDYFPVI + +++ ++   7 VQQWSVAVF+++   8 PFLPPAACFF +++   9 RILRFPWQL +++ ++  10 VWSDVTPLNF ++ +++  11YYSKSVGFMQW ++  12 STIRGELFFF ++ ++  13 HYTYILEVF ++ ++  14 SYSSCYSF ++ 15 KYALLLQDL + ++  16 TYNPDFSSL ++  17 YYADKKTFIVL + ++ +  18DYIGSVEKW + +  19 ILKEDPFLF +  20 EFTTVLYNF +  21 SYEVRSTF + + +  22TQPGDWTLF +  23 KFIISDWRF +  24 MYPDLSELLM + +  25 SYNGYVFYL + +  26KTPTNYYLF +  27 NYTLYPITF +  28 YYSIISHTL + +  29 VYPLLSRLYW + +  30QYLPGWTVLF +  31 QYQNVLTLW +  32 SLPDLTPTF + +  33 KSSVIASLLF +  34MQPRMFFLF + +  35 KYLEESVWL +  36 KQMEDGHTLF +  37 QWPWQASLQF +  38KYTNWKAFL +  39 LIFMLANVF +  40 QYEPPSAPSTTF +  41 VIYFMGAIF +  42TLPNTIYRF + +  43 IQMDEPMAF +  44 AYLSAVGTF +  45 KYFVPPQLF +  46AFPVTSIFHTF + +  47 KYADYFLEV +  48 VFIDHPVHLKF +  49 LYISEVRNI +  50SYPELVKMVW +  51 KYALLLQEL +  52 KYMKIFHKF +  53 KYITNLEDL +  54LLIKLLQTF +  55 RWMDQRLVF +  56 VYMIEPLEL +  57 YPSIIQEF +  84 YVDINTFRL+++ +++ + +++  85 YIDEFQSLV ++  86 FVIDGFDEL ++ ++ ++  87 TLYPYQISQL ++++ + ++  88 VQMVITEAQKV ++ ++  89 ILSTTMVTV ++ ++ + +  90 FLLMHPSI ++ 91 FALPGLLHA ++ ++  92 NLRDLLSEV ++ ++ + +  93 TLQEKILQV ++  94VLPDIETLIGV ++ ++  95 ITIGVLARV ++  96 HLVGGLHTV +  97 VLALVNSTV +  98LQSSGLTLLL + + +  99 FLKEKVPGI + 100 RQYPTPFQL + + 101 FIISDWRFVL + 102SLLEQAIAL + 103 FLYYPDPVL + 104 GMLDIFWGV + 105 SLLTHIPTA + 106FIIDTTYPAYV + 107 LLQGAIESV + 108 MIIALSLYI + + 109 LLLGSIGLLGV + 110LLADFQALL + 111 ALCLLLHLL + 112 SVSDGIHSV + 113 AVLTGLVEV + 114ILDERQVLL + + 115 MLLETQDALYV + + 116 VLMEENSKL + 117 FLDPNARPLV + 118ALSSVLHSI + 119 RTADITVTV + + 120 ALLANLPAV + + 121 ALVDTLTGI + 122ALLEMFPEITV + 123 LMAFFLAVV + 124 SVASVLLYL + 138 DSDSCHFNY ++ 139ECDMAFHIY + 140 ESDREELNY + 143 FIDYPKKEDY ++ + 146 GLNTGSALSY ++ + 147GSSDSSTLPKL + 148 GTEFTTILY + 149 GTEFTTVLY + 150 GTELLSLVY + 153KLDRSVFTAY ++ 155 LLDPNPHMY + ++ 158 LSDLLKQGY ++ + 160 LTEAVLNRY ++++ + ++ 163 NSDSSLTLREFY + + 164 NTDNNLAVY +++ +++ + 165 NTDPTAPPY + +166 NTQITDIGRY + + 167 QSDPGTSVLGY + 168 QTDHPQPILDRY + ++ 171RSDPVTLNVLY ++ 172 RTDSCSSAQAQY + 174 SADDIRGIQSLY +++ +++ + +++ 175SDVTPLTF +++ ++ 176 SRTINVSNLY + + 177 SSDEVNFLVY + + 178 SSDSSTLPKL +179 STAKSATVVTY ++ 183 TLEDIATSHLY + 185 TSDSNLNKY ++ 188 VSDAKLDKY +189 VSDSECLSRY +++ +++ 190 VTDGINPLIDRY ++ 192 VTEESFDSKFY + 193VTEFSLNTY ++ + 194 VVADTKMIEY ++ + 195 VVDSVGGYLY + ++ 199 YLPQHTIETY +200 YSDEDVTKY + ++ 201 YVGKEHMFY +++ +++ 202 KLAELEGALQK +++ + 203KVKDTPGLGK ++ ++ + + 204 AVFDKFIRY ++ 205 SLDGAARPK + + ++ 206KLIDLSQVMY + 207 RSFNGLLTMY + + 208 GLASRILDAK + + 209 RTQIPMSEK + 210ATSGVPVYK + + 211 TVNPVAIHK + 212 KAYEQVMHY + 213 LNINMTSPMGTK + 214RTMSEAALVRK + 215 MMFSGPQILKL + 216 KLYAWELAF + 217 RILNQILYY + 218KTLVAELLILK + + 219 RLRSSLVFK + 220 SPSVSQLSVL ++ +++ +++ 221 VPDVAQFVL+++ +++ 222 NPFYPEVEL +++ +++ 223 YPKDIYSSF ++ +++ 224 GPQPWHAAL +++++ + 225 LPFDGPGGIL +++ ++ 226 SPRMSGLLSQT +++ 227 YPRGNHWAVGH +++ 228YPRGNHWAVGHL +++ 229 VPLPAGGGTV +++ 230 VPLPAGGGTVL +++ 231 RPRALRDLQL +++ + 232 RPRALRDLQLL + ++ + 233 KPYQGNPTF ++ 234 RAKNAGVTI ++ ++ 235MPLKHYLLL ++ 236 RVRGGEDGDRAL ++ 237 RPAATAVISL ++ ++ 238 KPGPPWAAF ++239 YVPSASLFML + ++ 240 SPREVTTVL ++ 241 SARLATDAL ++ 242 SPRWLPVSL ++243 RPIENRILIL + ++ 244 FPYVRDFVM ++ + 245 RIREHVPQL ++ + 246TPLPAVIVL + ++ 247 RALLARLLL ++ + 248 IPNWARQDL ++ + 249 VPSSRILQL ++ +250 SPRDFLSGL ++ 251 VPRSSGQTV + + ++ 252 SPDIRNTTV ++ 253 RVIDAVRFTL ++254 NPFPHLITL + + + 255 MPLLENLYL + + 256 SPRVPSIEL + 257 LPRIPFADV + +++ 258 LPRGPLASL + + 259 RPPAAGLRGISL + 260 YPQHPGLNA + + 261APSARVGVC + + + 262 SAYPQRLEI + 263 HPAPYGDLL + + 264 RPILIIITL + 265SPRQPPRLV + 266 HAYPPGPGL + 267 HPELVNHIVF + 268 YPLFRGINL + + 269APRAPRLML + 270 APGPRFLVT + + 271 MPLPWSLALP + 272 MPLPWSLALPL + 273MPLLWLRGF + + + 274 TPYQEHVAL + 275 APHPPLSVV + 276 LPRAGGAFL + + 277MPLFEPRVF + + + 278 HPMIDINGIIVF + + 279 SPARASPAL + 280 VPISEEGTPVL +281 RPRAPVTPA + 282 MPQIETRVIL + + 283 RPHSLSSEL + + 284 FPVTSIFHTF + +285 FPSFLTNSL + 286 VPTLRSEL + 287 APREEQQRSL + 288 FPQKFIDLL + 289VPENHSVAL + 290 APYRPPDISL + 296 MPMQDIKM +++ +++ +++ 297 RAQLKLVAL +++298 FNKRKPLSL + ++ + 299 MAQFKEISL + ++ + 300 VASPKHCVL ++ ++ + + 301YMHKLLVL ++ 302 HLLQKQTSI ++ 303 LPFPKFTV ++ 304 ELKKLYCQI + 305ALKLRVAVL + 306 ILKVKVGL + 307 ILLPRTVSL + + 308 MLKQKVEEL + 309DAIQRKYSC + 310 LPPKKFVL + 311 EIRIRVVQM + 312 EAMLRNKEL + 313ELKKKEYEEL + 314 AIISRLVAL + 319 AEMLERVIKNY ++ +++ +++ 320 MEVDPIGHVYIF+++ +++ +++ 321 AEMLESVIKNY + +++ +++ 322 KEVDPAGHSY +++ +++ 323SEFMQVIF +++ +++ 324 TDSIHAWTF +++ 325 QEQDVDLVQKY +++ +++ 326 QEMQHFLGL+++ +++ +++ 327 YEIEARNQVF +++ +++ +++ 328 FEYDFLLQRI ++ +++ +++ 329NEHPSNNW ++ +++ 330 KEGDLGGKQW ++ ++ + + 331 EDAQGHIW ++ ++ 332MEVPVIKI + ++ + ++ 333 AETLSTIQI ++ ++ + 334 AEDEPAAAHL ++ ++ + + 335KELEATKQY ++ ++ + 336 ASSSGPMRVWV ++ ++ 337 TENRYCVQL ++ 338SEGSEPALLHSW ++ 339 SEPALLHSW ++ 340 TEFSLNTY ++ + 341 EEIEGKGSFTYF ++342 HEFSSPSHL ++ 343 TEFTTVLY + 344 EEATGQFHVY + 345 IEFIHPQAF + + 346VEAPGPVHVYW + + + 347 ALNPYQYQY + 348 AEIQGNINHV + 349 AEQDMRELTY + 350GECDVFKEIL + 351 EEVNYINTF + 352 NEVLTYIKF + 353 GEIIMQNNW + 354TEDPTILRI + 355 SDMVRFHLF + + + 356 EEGRVYLF + + 357 RELENCFQIQ + 358KEADIHFLI + + + 359 DELFSIALY + 360 AEVPTGVII + 361 SENLFFASF + 362SEKGVIQVY + 363 AELDKLTSV + 364 AETPIQNVI + 365 SEMNVNMKY + 366AENLFRAF + + 367 GEVHPSEMI + + 368 GEFPVRVQV + 369 EEIERFFKL + 370YEDLSQKY + 371 GELALKKKI + 372 TEGIIMKDF + 373 MEMQKSPVF + 374DEVNFLVY + + 375 VYSDLHAFYY ++ 376 KYVKDFHKF + 377 VYVGAVNRI + 378KFLGPAEHLTF + 388 FSIPEGALVAV + 389 TLMEQPLTTL + 401 DSDESYMEKSLY + 402DTDSQRLAY + 405 ETEEGIYWRY ++ 409 FVDPLVTNY + 419 LSELKPMSY + 427NSDEQKITEMVY + 429 NTEDSSMSGYLY + 432 NVDPVQHTY + 438 STDNFNCKY + + 442TSDFSRFTNY + 450 YSDDGQKWTVY ++ 454 KVYTPSISK + 455 RIADIFVKK + + 456SMFTAILKK + 457 SINKPTSER + 458 GIADFVLKY + 459 RPMQQARAQL +++ 460MPMAGDMNGL ++ 461 RPILIIVTL + 462 RPFHTRATV ++ ++ + + 463 TPKAGPTL ++++ + + 464 YPRPGTPAA + 465 VPRPIFSQL + 466 APYKSVTSL + + 467 KPFSSFTSM +468 SPMYGQAGL + 469 YPENGVVQM + 470 SPNSYFRVL + 471 KPRPDVTNEL + 472NPRATDAQL + 476 AEEEIMKKI +++ +++ + +++ 477 QENSYQSRL ++ +++ 478SEIEQEIGSL ++ ++ 479 AEIQPQTQV + + + 480 GEVSGLTKDF + 481 RELQHEHSL + +482 TEREWADEW + 483 EENDQSTHKW + 484 AEVGFVRFF + 485 SEIEDSTKQVF + 486SEDDPILQI + 487 AEDQLHHSF + 488 TEFPIIKMY + 489 SEIGKAVGF +

Example 3

In Vitro Immunogenicity for MHC Class I Presented Peptides

In order to obtain information regarding the immunogenicity of theTUMAPs of the present invention, the inventors performed investigationsusing an in vitro T-cell priming assay based on repeated stimulations ofCD8+ T cells with artificial antigen presenting cells (aAPCs) loadedwith peptide/MHC complexes and anti-CD28 antibody. This way theinventors could show immunogenicity for for HLA-A*02:01, HLA-A*24:02,HLA-A*01:01, HLA-A*03:01, HLA-B*07:02, HLA-B*08:01 and HLA-B*44:02restricted TUMAPs of the invention, demonstrating that these peptidesare T-cell epitopes against which CD8+ precursor T cells exist in humans(Table 10a and Table 10b).

In Vitro Priming of CD8+ T Cells

In order to perform in vitro stimulations by artificial antigenpresenting cells loaded with peptide-MHC complex (pMHC) and anti-CD28antibody, the inventors first isolated CD8+ T cells from fresh HLA-A*02,HLA-A*24, HLA-A*01, HLA-A*03, HLA-B*07, HLA-B*08 or HLA-B*44leukapheresis products via positive selection using CD8 microbeads(Miltenyi Biotec, Bergisch-Gladbach, Germany) of healthy donors obtainedfrom the University clinics Mannheim, Germany, after informed consent.

PBMCs and isolated CD8+ lymphocytes were incubated in T-cell medium(TCM) until use consisting of RPMI-Glutamax (Invitrogen, Karlsruhe,Germany) supplemented with 10% heat inactivated human AB serum(PAN-Biotech, Aidenbach, Germany), 100 U/ml Penicillin/100 μg/mlStreptomycin (Cambrex, Cologne, Germany), 1 mM sodium pyruvate (CC Pro,Oberdorla, Germany), 20 μg/ml Gentamycin (Cambrex). 2.5 ng/ml IL-7(PromoCell, Heidelberg, Germany) and 10 U/ml IL-2 (Novartis Pharma,Nirnberg, Germany) were also added to the TCM at this step.

Generation of pMHC/anti-CD28 coated beads, T-cell stimulations andreadout was performed in a highly defined in vitro system using fourdifferent pMHC molecules per stimulation condition and 8 different pMHCmolecules per readout condition.

The purified co-stimulatory mouse IgG2a anti human CD28 Ab 9.3 (Jung etal., 1987) was chemically biotinylated usingSulfo-N-hydroxysuccinimidobiotin as recommended by the manufacturer(Perbio, Bonn, Germany). Beads used were 5.6 μm diameter streptavidincoated polystyrene particles (Bangs Laboratories, Illinois, USA).

pMHC used for positive and negative control stimulations wereA*02:01/MLA-001 (peptide ELAGIGILTV (SEQ ID NO. 532) from modifiedMelan-A/MART-1) and A*02:01/DDX5-001 (YLLPAIVHI from DDX5, SEQ ID NO.533), respectively.

800.000 beads/200 μl were coated in 96-well plates in the presence of4×12.5 ng different biotin-pMHC, washed and 600 ng biotin anti-CD28 wereadded subsequently in a volume of 200 μl. Stimulations were initiated in96-well plates by co-incubating 1×10⁶ CD8+ T cells with 2×10⁵ washedcoated beads in 200 μl TCM supplemented with 5 ng/ml IL-12 (PromoCell)for 3 days at 37° C. Half of the medium was then exchanged by fresh TCMsupplemented with 80 U/ml IL-2 and incubating was continued for 4 daysat 37° C. This stimulation cycle was performed for a total of threetimes. For the pMHC multimer readout using 8 different pMHC moleculesper condition, a two-dimensional combinatorial coding approach was usedas previously described (Andersen et al., 2012) with minor modificationsencompassing coupling to 5 different fluorochromes. Finally, multimericanalyses were performed by staining the cells with Live/dead near IR dye(Invitrogen, Karlsruhe, Germany), CD8-FITC antibody clone SK1 (BD,Heidelberg, Germany) and fluorescent pMHC multimers. For analysis, a BDLSRII SORP cytometer equipped with appropriate lasers and filters wasused. Peptide specific cells were calculated as percentage of total CD8+cells. Evaluation of multimeric analysis was done using the FlowJosoftware (Tree Star, Oregon, USA). In vitro priming of specificmultimer+CD8+ lymphocytes was detected by comparing to negative controlstimulations. Immunogenicity for a given antigen was detected if atleast one evaluable in vitro stimulated well of one healthy donor wasfound to contain a specific CD8+ T-cell line after in vitro stimulation(i.e. this well contained at least 1% of specific multimer+ among CD8+T-cells and the percentage of specific multimer+ cells was at least 10×the median of the negative control stimulations).

In Vitro Immunogenicity for Lung Cancer (Including NSCLC and SCLC)Peptides

For tested HLA class I peptides, in vitro immunogenicity could bedemonstrated by generation of peptide specific T-cell lines. Exemplaryflow cytometry results after TUMAP-specific multimer staining for 16peptides of the invention are shown in FIGS. 3A through 11B togetherwith corresponding negative controls. Results for 152 peptides from theinvention are summarized in Table 10a and Table 10b.

TABLE 10a  in vitro immunogenicity of HLA class I peptides ofthe invention Exemplary results of in vitro immunogenicityexperiments conducted by the applicant for thepeptides of the invention. <20% = +;  20% - 49% = ++; 50% - 69% =+++; >= 70% = ++++ Wells positive Seq ID No Sequence [%] 491 KYLEKYYNL++ 492 NYEDHFPLL ++ 493 TYKYVDINTF + 494 RYLEKFYGL + 495 SYNDALLTF +++496 VFMKDGFFYF + 498 EYIRALQQL + 500 VWSDVTPLTF + 504 VYEKNGYIYF ++++510 KVLEHVVRV + 513 KLVELEHTL + 515 KIFEMLEGV + 516 YTFSGDVQL + 519KIQEILTQV + 520 KIQEMQHFL + 525 RLDDLKMTV + 528 RLLDSVSRL +

TABLE 10b in vitro immunogenicity of HLA class I peptides ofthe invention Exemplary results of in vitro immunogenicityexperiments conducted by the applicant for thepeptides of the invention. <20% = +; 20% - 49% = ++; 50% - 69% = +++; >=70% ++++ Seq ID No Sequence Wells positive [%] HLA 136 ATDLVVLDRY “+”A*01 143 FIDYPKKEDY “+” A*01 153 KLDRSVFTAY “++++” A*01 160 LTEAVLNRY“++” A*01 164 NTDNNLAVY “+” A*01 173 RTEFNLNQY “++++” A*01 174SADDIRGIQSLY “+” A*01 185 TSDSNLNKY “+” A*01 187 VADLHLYLY “+++” A*01189 VSDSECLSRY “++” A*01 193 VTEFSLNTY “+” A*01 201 YVGKEHMFY “+” A*01395 ASEASRLAHY “++” A*01 398 ASEQQALHTVQY “+” A*01 405 ETEEGIYWRY “+”A*01 430 NTEGLHHLY “+” A*01 436 QTETGTPYMLY “+” A*01 451 YSDSLVQKGY “+”A*01 452 YVDAVLGKGHQY “+” A*01 84 YVDINTFRL “+” A*02 85 YIDEFQSLV “++”A*02 87 TLYPYQISQL “++” A*02 88 VQMVITEAQKV “++” A*02 89 ILSTTMVTV “+++”A*02 93 TLQEKILQV “+” A*02 94 VLPDIETLIGV “+++” A*02 95 ITIGVLARV “++++”A*02 96 HLVGGLHTV “+++” A*02 97 VLALVNSTV “+++” A*02 101 FIISDWRFVL “+”A*02 125 VLQPFLPSI “++++” A*02 127 GLDGSLVFL “++” A*02 128 FLGTTPTL “+”A*02 129 VLYDKDAVYV “++” A*02 130 NLWGGQGLLGV “+” A*02 131 LLKEFVQRV“++++” A*02 132 ALWLVDPLTV “+++” A*02 133 MTLPVDAVISV “+” A*02 392SLFKDQMEL “+” A*02 393 ILLPYLQTL “+” A*02 205 SLDGAARPK “++” A*03 208GLASRILDAK “+” A*03 209 RTQIPMSEK “+” A*03 210 ATSGVPVYK “++++” A*03 214RTMSEAALVRK “+” A*03 218 KTLVAELLILK “+” A*03 1 QYDPTPLTW “+” A*24 2VWSNVTPLKF “+” A*24 4 SYEKVINYL “+” A*24 6 KYKDYFPVI “+” A*24 8PFLPPAACFF “+” A*24 10 VWSDVTPLNF “+” A*24 11 YYSKSVGFMQW “++” A*24 13HYTYILEVF “+++” A*24 15 KYALLLQDL “+++” A*24 16 TYNPDFSSL “+” A*24 59KYSTTFFMV “++” A*24 60 TYLSIFDQL “+” A*24 61 NYAENILTL “++” A*24 62LYQEILAQL “+” A*24 65 VYPASKMFPFI “+” A*24 66 IYFRDSSFL “++” A*24 72RYEGILYTI “+” A*24 76 WYGWHFPEL “+” A*24 79 RYLADLPTL “+” A*24 83TYCQNIKEF “+” A*24 375 VYSDLHAFYY “+” A*24 379 NYIVPDKQIF “+” A*24 380VFQEKHHVI “+” A*24 383 RYKQDVERF “+++” A*24 384 KYVKVFDKF “+++” A*24 386VYNDHSIYVW “++” A*24 220 SPSVSQLSVL “++++” B*07 221 VPDVAQFVL “++” B*07222 NPFYPEVEL “+” B*07 223 YPKDIYSSF “++” B*07 224 GPQPWHAAL “++” B*07225 LPFDGPGGIL “++++” B*07 226 SPRMSGLLSQT “+++” B*07 228 YPRGNHWAVGHL“++” B*07 231 RPRALRDLQL “++” B*07 232 RPRALRDLQLL “+++” B*07 233KPYQGNPTF “+” B*07 237 RPAATAVISL “+” B*07 241 SARLATDAL “+++” B*07 242SPRWLPVSL “++++” B*07 244 FPYVRDFVM “+” B*07 245 RIREHVPQL “++” B*07 248IPNWARQDL “++++” B*07 249 VPSSRILQL “+++” B*07 250 SPRDFLSGL “+” B*07252 SPDIRNTTV “+” B*07 274 TPYQEHVAL “+” B*07 285 FPSFLTNSL “++++” B*07292 SPQRLRGLLL “+++” B*07 293 RPRSALPRLLLP “++” B*07 294 GPTPNTGAAL“+++” B*07 460 MPMAGDMNGL “++” B*07 462 RPFHTRATV “++” B*07 463 TPKAGPTL“+” B*07 473 LPRALLSSL “++” B*07 474 LPRLLPAL “+++” B*07 320MEVDPIGHVYIF “+” B*44 322 KEVDPAGHSY “++” B*44 323 SEFMQVIF “+” B*44 325QEQDVDLVQKY “+” B*44 326 QEMQHFLGL “+” B*44 328 FEYDFLLQRI “+” B*44 329NEHPSNNW “+” B*44 330 KEGDLGGKQW “+” B*44 331 EDAQGHIW “++” B*44 333AETLSTIQI “+” B*44 334 AEDEPAAAHL “+” B*44 337 TENRYCVQL “++” B*44 338SEGSEPALLHSW “+” B*44 339 SEPALLHSW “++” B*44 342 HEFSSPSHL “+” B*44 476AEEEIMKKI “+” B*44 477 QENSYQSRL “+” B*44 297 RAQLKLVAL “+” B*08 298FNKRKPLSL “+” B*08 299 MAQFKEISL “++++” B*08 300 VASPKHCVL “+” B*08 303LPFPKFTV “++” B*08 305 ALKLRVAVL “+” B*08 306 ILKVKVGL “+” B*08 307ILLPRTVSL “+” B*08 308 MLKQKVEEL “+” B*08 311 EIRIRVVQM “+” B*08 312EAMLRNKEL “+” B*08 313 ELKKKEYEEL “+” B*08 314 AIISRLVAL “++” B*08 315DIYQRALNL “+” B*08 316 VIKEKALTL “+” B*08 318 EAAIRSVEL “++” B*08

Example 4

Synthesis of Peptides

All peptides were synthesized using standard and well-established solidphase peptide synthesis using the Fmoc-strategy. Identity and purity ofeach individual peptide have been determined by mass spectrometry andanalytical RP-HPLC. The peptides were obtained as white to off-whitelyophilizes (trifluoro acetate salt) in purities of >50%. All TUMAPs arepreferably administered as trifluoro-acetate salts or acetate salts,other salt-forms are also possible.

Example 5

MHC Binding Assays

Candidate peptides for T cell based therapies according to the presentinvention were further tested for their MHC binding capacity (affinity).The individual peptide-MHC complexes were produced by UV-ligandexchange, where a UV-sensitive peptide is cleaved upon UV-irradiation,and exchanged with the peptide of interest as analyzed. Only peptidecandidates that can effectively bind and stabilize the peptide-receptiveMHC molecules prevent dissociation of the MHC complexes. To determinethe yield of the exchange reaction, an ELISA was performed based on thedetection of the light chain (p2m) of stabilized MHC complexes. Theassay was performed as generally described in Rodenko et al. (Rodenko etal., 2006).

96 well MAXISorp plates (NUNC) were coated over night with 2 ug/mlstreptavidin in PBS at room temperature, washed 4× and blocked for 1 hat 37° C. in 2% BSA containing blocking buffer. RefoldedHLA-A*02:01/MLA-001 monomers served as standards, covering the range of15-500 ng/ml. Peptide-MHC monomers of the UV-exchange reaction werediluted 100-fold in blocking buffer. Samples were incubated for 1 h at37° C., washed four times, incubated with 2 ug/ml HRP conjugatedanti-β2m for 1 h at 37° C., washed again and detected with TMB solutionthat is stopped with NH₂SO₄. Absorption was measured at 450 nm.Candidate peptides that show a high exchange yield (preferably higherthan 50%, most preferred higher than 75%) are generally preferred for ageneration and production of antibodies or fragments thereof, and/or Tcell receptors or fragments thereof, as they show sufficient avidity tothe MHC molecules and prevent dissociation of the MHC complexes.

TABLE 11 MHC class I binding scores. Binding of HLA-class Irestricted peptides to HLA-A*01 was ranged by pep-tide exchange yield: >10% = +; >20% = ++; >50 = +++; >75% = ++++Seq ID No Sequence Peptide exchange 134 AAEIGDKSWLY “++++” 135 ASEDSVLLY“++++” 136 ATDLVVLDRY “++++” 137 ATSKFMEFY “++++” 138 DSDSCHFNY “++++”139 ECDMAFHIY “++++” 140 ESDREELNY “+++” 141 ESDVGVVVY “+++” 142EVAEPSVLFDLY “+++” 143 FIDYPKKEDY “+++” 144 FLDSQNLSAY “+++” 145FVDKPVAY “+++” 146 GLNTGSALSY “++” 147 GSSDSSTLPKL “++” 148 GTEFTTILY“++++” 149 GTEFTTVLY “++++” 150 GTELLSLVY “++++” 151 HSDLKVGEY “+++” 152HTDSLHLLI “+++” 153 KLDRSVFTAY “+++” 154 LLDISQKNLY “+++” 155 LLDPNPHMY“++++” 156 LLDSLREQY “+++” 157 LMDRPIFY “++++” 158 LSDLLKQGY “++++” 159LSDTSVIQFY “++++” 160 LTEAVLNRY “+++” 161 LVDDGTHGQY “+++” 162LVDNSIRELQY “+++” 163 NSDSSLTLREFY “+++” 164 NTDNNLAVY “++++” 165NTDPTAPPY “+++” 166 NTQITDIGRY “+++” 167 QSDPGTSVLGY “+++” 168QTDHPQPILDRY “++++” 169 RLDTPLYFSY “++++” 170 RSDDTAVYY “++++” 171RSDPVTLNVLY “++++” 172 RTDSCSSAQAQY “+++” 173 RTEFNLNQY “++++” 174SADDIRGIQSLY “++++” 176 SRTINVSNLY “+++” 177 SSDEVNFLVY “++++” 178SSDSSTLPKL “++” 179 STAKSATWTY “++++” 180 STDPWIQMAY “++++” 181TADGKTYYY “+++” 182 TDYHVRVY “+++” 183 TLEDIATSHLY “++++” 184TSAHPEDSSFY “+++” 185 TSDSNLNKY “++++” 186 TTDIIEKY “+++” 187 VADLHLYLY“+++” 188 VSDAKLDKY “+++” 189 VSDSECLSRY “++++” 190 VTDGINPLIDRY “+++”191 VTDGSLYEGVAY “++++” 192 VTEESFDSKFY “++++” 193 VTEFSLNTY “++++” 194VVADTKMIEY “+” 195 VVDSVGGYLY “++++” 196 WMFFVINY “+” 197 YADTVRPEFY“+++” 198 YLDPVQRDLY “++++” 199 YLPQHTIETY “++” 200 YSDEDVTKY “++++” 201YVGKEHMFY “++++” 394 ASEAEMRLFY “++++” 395 ASEASRLAHY “++++” 396ASEFGNHYLY “++++” 397 ASEITSKGASLY “++++” 398 ASEQQALHTVQY “++++” 399ATDIPCLLY “++++” 400 ATDISRQNEY “+++” 401 DSDESYMEKSLY “++++” 402DTDSQRLAY “+++” 403 ELDSKVEVLTY “+++” 404 ETARKFLYY “++++” 405ETEEGIYWRY “++++” 406 ETEQTKFWDY “++++” 407 FSDNDKLYLY “++++” 408FTEQWTDGY “+++” 409 FVDPLVTNY “++++” 410 GSDHQSPSSSSY “++++” 411GTVYEDLRY “++++” 412 ILDEVIMGY “++++” 413 ISDRYYTALY “++++” 414KTDESLTKY “+++” 415 LLDPRSYHTY “+++” 416 LLDTAQKNLY “++++” 417LLEDKHFQSY “++++” 418 LSDPSGPKSY “+++” 419 LSELKPMSY “++++” 420LTEDKETLQY “++++” 421 LTELLERAAFY “++++” 422 MIDVTKSYY “++++” 423NLDAVHDITVAY “++++” 424 NLDEEKQLLY “+++” 425 NLDIIQQEY “++++” 426NLDQATRVAY “+++” 427 NSDEQKITEMVY “++++” 428 NSELSCQLY “++++” 429NTEDSSMSGYLY “++++” 430 NTEGLHHLY “++++” 431 NTSDMMGRMSY “++++” 432NVDPVQHTY “+++” 433 QIDTGENLY “++++” 434 QTDCAPNNGY “++++” 435QTDDTWRTEY “++++” 436 QTETGTPYMLY “++++” 437 STDGKHWWEY “++++” 438STDNFNCKY “+++” 439 TLDAGKFQIY “+++” 440 TLDENPGVRY “+++” 441TLDSALNAASYY “++++” 442 TSDFSRFTNY “++++” 443 TTDFPSESSFEY “++++” 444TTDTVIRSY “+++” 445 VLDQGKITEY “+++” 446 VTAQVVGTERY “++++” 447VVDEDHELIY “+++” 448 YLDIPNPRY “+++” 449 YLDRGTGNVSFY “++++” 450YSDDGQKWTVY “++++” 451 YSDSLVQKGY “++++” 452 YVDAVLGKGHQY “++++”

TABLE 12 MHC class I binding scores. Binding of HLA-class Irestricted peptides to HLA-A*02 was ranged bypeptide exchange yield: >10% = +; >20% = ++; >50 = +++; >75% = ++++Seq ID No Sequence Peptide exchange 84 YVDINTFRL “++++” 85 YIDEFQSLV“++++” 86 FVIDGFDEL “++++” 87 TLYPYQISQL “++++” 88 VQMVITEAQKV “++++” 89ILSTTMVTV “++++” 91 FALPGLLHA “+++” 92 NLRDLLSEV “+++” 93 TLQEKILQV“++++” 94 VLPDIETLIGV “++++” 95 ITIGVLARV “++++” 96 HLVGGLHTV “++++” 97VLALVNSTV “++++” 98 LQSSGLTLLL “++++” 99 FLKEKVPGI “++++” 100 RQYPTPFQL“++++” 101 FIISDWRFVL “+++” 102 SLLEQAIAL “++++” 103 FLYYPDPVL “++++”104 GMLDIFWGV “++++” 105 SLLTHIPTA “++++” 106 FIIDTTYPAYV “++++” 107LLQGAIESV “++++” 109 LLLGSIGLLGV “++++” 110 LLADFQALL “++++” 111ALCLLLHLL “+” 112 SVSDGIHSV “++++” 113 AVLTGLVEV “++++” 114 ILDERQVLL“++++” 115 MLLETQDALYV “++++” 116 VLMEENSKL “++++” 117 FLDPNARPLV “++++”118 ALSSVLHSI “++++” 119 RTADITVTV “++++” 120 ALLANLPAV “++++” 121ALVDTLTGI “++++” 122 ALLEMFPEITV “++++” 123 LMAFFLAVV “++” 124 SVASVLLYL“++++” 125 VLQPFLPSI “++++” 126 FLSTVTSV “++++” 127 GLDGSLVFL “++++” 128FLGTTPTL “++++” 129 VLYDKDAVYV “++++” 130 NLWGGQGLLGV “++++” 131LLKEFVQRV “++++” 132 ALWLVDPLTV “++++” 133 MTLPVDAVISV “++++” 388FSIPEGALVAV “++++” 389 TLMEQPLTTL “++++” 390 HIMPTVHTV “++++” 391SLIDMRGIETV “++++” 392 SLFKDQMEL “++++” 393 ILLPYLQTL “++++”

TABLE 13 MHC class I binding scores. Binding of HLA-class Irestricted peptides to HLA-A*03 was ranged bypeptide exchange yield: >10% = +; >20% = ++; >50 = +++; >75% = ++++Seq ID No Sequence Peptide exchange 202 KLAELEGALQK “++++” 203KVKDTPGLGK “++++” 204 AVFDKFIRY “++++” 205 SLDGAARPK “++++” 206KLIDLSQVMY “++++” 207 RSFNGLLTMY “++++” 208 GLASRILDAK “++++” 209RTQIPMSEK “++++” 210 ATSGVPVYK “++++” 211 TVNPVAIHK “++++” 212 KAYEQVMHY“++++” 214 RTMSEAALVRK “++++” 215 MMFSGPQILKL “++++” 216 KLYAWELAF“++++” 217 RILNQILYY “+++” 218 KTLVAELLILK “++++” 219 RLRSSLVFK “+++”453 AINTSIKNK “++++” 454 KVYTPSISK “++++” 455 RIADIFVKK “++++” 456SMFTAILKK “++++” 457 SINKPTSER “++++” 458 GIADFVLKY “++++”

TABLE 14 MHC class I binding scores. Binding of HLA-class Irestricted peptides to HLA-A*24 was ranged bypeptide exchange yield: >10% = +; >20% = ++; >50 = +++; >75% = ++++Seq ID No Sequence Peptide exchange 1 QYDPTPLTW “++++” 2 VWSNVTPLKF“++++” 3 YLEKFYGL “++” 4 SYEKVINYL “++++” 5 RYMKKDYLI “++++” 6 KYKDYFPVI“++++” 7 VQQWSVAVF “++++” 8 PFLPPAACFF “++++” 10 VWSDVTPLNF “++++” 11YYSKSVGFMQW “++++” 12 STIRGELFFF “+” 13 HYTYILEVF “++++” 14 SYSSCYSF“++++” 15 KYALLLQDL “++++” 16 TYNPDFSSL “+++” 17 YYADKKTFIVL “+++” 18DYIGSVEKW “++++” 19 ILKEDPFLF “+” 20 EFTTVLYNF “++++” 21 SYEVRSTF “+++”22 TQPGDWTLF “++++” 23 KFIISDWRF “++++” 24 MYPDLSELLM “++++” 25SYNGYVFYL “++++” 26 KTPTNYYLF “++++” 27 NYTLYPITF “++++” 28 YYSIISHTL“++++” 29 VYPLLSRLYW “++++” 30 QYLPGWTVLF “++++” 31 QYQNVLTLW “++++” 32SLPDLTPTF “++++” 33 KSSVIASLLF “+++” 34 MQPRMFFLF “++++” 35 KYLEESVWL“++++” 36 KQMEDGHTLF “++” 37 QWPWQASLQF “++++” 38 KYTNWKAFL “++++” 39LIFMLANVF “+” 40 QYEPPSAPSTTF “+++” 42 TLPNTIYRF “++++” 43 IQMDEPMAF “+”44 AYLSAVGTF “++++” 45 KYFVPPQLF “++++” 46 AFPVTSIFHTF “++++” 47KYADYFLEV “++++” 48 VFIDHPVHLKF “++++” 49 LYISEVRNI “++++” 50 SYPELVKMVW“++++” 51 KYALLLQEL “++++” 52 KYMKIFHKF “++++” 53 KYITNLEDL “++++” 54LLIKLLQTF “+++” 55 RWMDQRLVF “++++” 56 VYMIEPLEL “++++” 57 YPSIIQEF“++++” 58 QFAAPLRGIYF “++++” 59 KYSTTFFMV “++” 60 TYLSIFDQL “+++” 61NYAENILTL “+++” 62 LYQEILAQL “++++” 63 VMPSDSFFF “++++” 64 NYAIFDEGHML“++++” 65 VYPASKMFPFI “++++” 66 IYFRDSSFL “++++” 67 RYPGKFYRV “++++” 68IYQQIIQTY “++++” 69 IMPEKFEFW “++++” 70 PYTNYTFDF “++++” 71 SYMVLAPVF“++++” 72 RYEGILYTI “++++” 73 SYIGLPLTL “+++” 74 VYDQYFITL “++++” 75NYIYSISVF “++++” 76 WYGWHFPEL “+++” 77 AYTLLGHEFV “+” 78 TWFPKTPMLF“++++” 79 RYLADLPTL “++++” 80 YYSPLRDLL “++++” 81 LYPEGLRLL “++++” 82RFLPSPVVI “++++” 83 TYCQNIKEF “++++” 375 VYSDLHAFYY “++++” 376 KYVKDFHKF“++++” 377 VYVGAVNRI “++++” 378 KFLGPAEHLTF “++++” 379 NYIVPDKQIF “+++”380 VFQEKHHVI “+++” 381 TYSKKHFRI “++++” 382 IYHSHHPTL “+++” 383RYKQDVERF “+++” 384 KYVKVFDKF “++++” 385 MYINEVERL “++++” 386 VYNDHSIYVW“++++” 387 RWLPQKNAAQF “+++”

TABLE 15 MHC class I binding scores. Binding of HLA-class Irestricted peptides to HLA-B*07 was ranged bypeptide exchange yield: >10% = +; >20% = ++; >50 = +++; >75% = ++++Seq ID No Sequence Peptide exchange 220 SPSVSQLSVL “++++” 221 VPDVAQFVL“++++” 222 NPFYPEVEL “+++” 223 YPKDIYSSF “++++” 224 GPQPWHAAL “++++” 225LPFDGPGGIL “++++” 226 SPRMSGLLSQT “++++” 227 YPRGNHWAVGH “++++” 228YPRGNHWAVGHL “++++” 229 VPLPAGGGTV “+++” 230 VPLPAGGGTVL “+++” 231RPRALRDLQL “++++” 232 RPRALRDLQLL “++++” 233 KPYQGNPTF “++++” 234RAKNAGVTI “++++” 235 MPLKHYLLL “++++” 236 RVRGGEDGDRAL “++++” 237RPAATAVISL “+++” 238 KPGPPWAAF “++++” 239 YVPSASLFML “++++” 240SPREVTTVL “++++” 241 SARLATDAL “++++” 242 SPRWLPVSL “++++” 243RPIENRILIL “++++” 244 FPYVRDFVM “++++” 245 RIREHVPQL “+++” 246 TPLPAVIVL“++++” 247 RALLARLLL “+++” 248 IPNWARQDL “++++” 249 VPSSRILQL “++++” 250SPRDFLSGL “++++” 251 VPRSSGQTV “++++” 252 SPDIRNTTV “++++” 253RVIDAVRFTL “+++” 254 NPFPHLITL “++++” 255 MPLLENLYL “++++” 256 SPRVPSIEL“++++” 257 LPRIPFADV “++++” 258 LPRGPLASL “++++” 259 RPPAAGLRGISL “++++”260 YPQHPGLNA “+++” 261 APSARVGVC “+++” 262 SAYPQRLEI “+” 263 HPAPYGDLL“++++” 265 SPRQPPRLV “++++” 267 HPELVNHIVF “++” 268 YPLFRGINL “++++” 269APRAPRLML “++++” 270 APGPRFLVT “++++” 271 MPLPWSLALP “+++” 272MPLPWSLALPL “++++” 273 MPLLWLRGF “++” 274 TPYQEHVAL “+++” 275 APHPPLSVV“+++” 276 LPRAGGAFL “+++” 278 HPMIDINGIIVF “++” 279 SPARASPAL “+++” 280VPISEEGTPVL “+++” 281 RPRAPVTPA “++++” 282 MPQIETRVIL “+++” 283RPHSLSSEL “++++” 284 FPVTSIFHTF “++” 285 FPSFLTNSL “++++” 286 VPTLRSEL“++++” 287 APREEQQRSL “+++” 288 FPQKFIDLL “++” 289 VPENHSVAL “++++” 290APYRPPDISL “++++” 291 SPQRLRGLL “++++” 292 SPQRLRGLLL “+++” 293RPRSALPRLLLP “++++” 294 GPTPNTGAAL “++++” 295 KPEGTRIAV “++++” 459RPMQQARAQL “+++” 460 MPMAGDMNGL “++++” 462 RPFHTRATV “++++” 463 TPKAGPTL“++++” 464 YPRPGTPAA “++++” 465 VPRPIFSQL “++++” 466 APYKSVTSL “++++”467 KPFSSFTSM “++++” 468 SPMYGQAGL “++++” 469 YPENGVVQM “++” 470SPNSYFRVL “++++” 471 KPRPDVTNEL “++++” 472 NPRATDAQL “++++” 473LPRALLSSL “++++” 474 LPRLLPAL “++++” 475 RPHKPGLYL “++++”

TABLE 16 MHC class I binding scores. Binding of HLA-class Irestricted peptides to HLA-B*08 was ranged bypeptide exchange yield: >10% = +; >20% = ++; >50 = +++; >75% = ++++Seq ID No Sequence Peptide exchange 296 MPMQDIKM “++++” 297 RAQLKLVAL“++++” 298 FNKRKPLSL “+++” 299 MAQFKEISL “++++” 300 VASPKHCVL “+++” 301YMHKLLVL “++++” 302 HLLQKQTSI “+++” 303 LPFPKFTV “++++” 304 ELKKLYCQI“++++” 305 ALKLRVAVL “++++” 306 ILKVKVGL “++++” 307 ILLPRTVSL “++++” 308MLKQKVEEL “++++” 309 DAIQRKYSC “+++” 310 LPPKKFVL “++++” 311 EIRIRVVQM“++++” 312 EAMLRNKEL “++++” 313 ELKKKEYEEL “++++” 314 AIISRLVAL “++++”315 DIYQRALNL “++++” 316 VIKEKALTL “+++” 317 LVKVKVLL “++++” 318EAAIRSVEL “++++”

TABLE 17 MHC class I binding scores. Binding of HLA-class Irestricted peptides to HLA-B*44 was ranged bypeptide exchange yield: >10% = +; >20% = ++; >50 = +++; >75% = ++++Seq ID No Sequence Peptide exchange 319 AEMLERVIKNY “++++” 320MEVDPIGHVYIF “++++” 321 AEMLESVIKNY “+++” 322 KEVDPAGHSY “++” 323SEFMQVIF “+++” 324 TDSIHAWTF “+++” 325 QEQDVDLVQKY “++” 326 QEMQHFLGL“+++” 327 YEIEARNQVF “+++” 328 FEYDFLLQRI “++++” 329 NEHPSNNW “+++” 330KEGDLGGKQW “+++” 331 EDAQGHIW “+++” 332 MEVPVIKI “++” 333 AETLSTIQI“+++” 334 AEDEPAAAHL “++” 335 KELEATKQY “++” 336 ASSSGPMRWW “++” 337TENRYCVQL “++++” 338 SEGSEPALLHSW “+++” 339 SEPALLHSW “+++” 340 TEFSLNTY— 341 EEIEGKGSFTYF “+++” 342 HEFSSPSHL “+++” 343 TEFTTVLY “++” 344EEATGQFHVY “+++” 345 IEFIHPQAF “++++” 346 VEAPGPVHVYW “++++” 347ALNPYQYQY “+++” 348 AEIQGNINHV “+++” 349 AEQDMRELTY “+++” 350 GECDVFKEIL“+++” 351 EEVNYINTF “+++” 352 NEVLTYIKF “++++” 353 GEIIMQNNW “++++” 354TEDPTILRI “+++” 355 SDMVRFHLF “++” 356 EEGRVYLF “+++” 357 RELENCFQIQ“+++” 358 KEADIHFLI “++++” 359 DELFSIALY “++++” 360 AEVPTGVII “+++” 361SENLFFASF “++++” 362 SEKGVIQVY “+++” 363 AELDKLTSV “+++” 364 AETPIQNVI“+++” 365 SEMNVNMKY “++++” 366 AENLFRAF “++” 367 GEVHPSEMI “+++” 368GEFPVRVQV “++” 369 EEIERFFKL “+++” 370 YEDLSQKY “+” 371 GELALKKKI “++”372 TEGIIMKDF “++” 373 MEMQKSPVF “+++” 374 DEVNFLVY “+” 476 AEEEIMKKI“++” 477 QENSYQSRL “+++” 478 SEIEQEIGSL “+++” 479 AEIQPQTQV “+++” 480GEVSGLTKDF “++” 481 RELQHEHSL “+++” 482 TEREWADEW “+++” 483 EENDQSTHKW“+++” 484 AEVGFVRFF “++++” 485 SEIEDSTKQVF “+++” 486 SEDDPILQI “+++” 487AEDQLHHSF “+++” 488 TEFPIIKMY “++” 489 SEIGKAVGF “++++”

REFERENCE LIST

-   Allison, J. P. et al., Science 270 (1995): 932-933-   Andersen, R. S. et al., Nat. Protoc. 7 (2012): 891-902-   Appay, V. et al., Eur. J Immunol. 36 (2006): 1805-1814-   Banchereau, J. et al., Cell 106 (2001): 271-274-   Beatty, G. et al., J Immunol 166 (2001): 2276-2282-   Beggs, J. D., Nature 275 (1978): 104-109-   Benjamini, Y. et al., Journal of the Royal Statistical Society.    Series B (Methodological), Vol. 57 (1995): 289-300-   Boulter, J. M. et al., Protein Eng 16 (2003): 707-711-   Braumuller, H. et al., Nature (2013)-   Brossart, P. et al., Blood 90 (1997): 1594-1599-   Bruckdorfer, T. et al., Curr. Pharm. Biotechnol. 5 (2004): 29-43-   Card, K. F. et al., Cancer Immunol Immunother. 53 (2004): 345-357-   Cohen, C. J. et al., J Mol Recognit. 16 (2003a): 324-332-   Cohen, C. J. et al., J Immunol 170 (2003b): 4349-4361-   Cohen, S. N. et al., Proc. Natl. Acad. Sci. U.S.A 69 (1972):    2110-2114-   Coligan, J. E. et al., Current Protocols in Protein Science (1995)-   Colombetti, S. et al., J Immunol. 176 (2006): 2730-2738-   Dengjel, J. et al., Clin Cancer Res 12 (2006): 4163-4170-   Denkberg, G. et al., J Immunol 171 (2003): 2197-2207-   Falk, K. et al., Nature 351 (1991): 290-296-   Follenzi, A. et al., Nat Genet. 25 (2000): 217-222-   Fong, L. et al., Proc. Natl. Acad. Sci. U.S.A 98 (2001): 8809-8814-   Gabrilovich, D. I. et al., Nat Med. 2 (1996): 1096-1103-   Gattinoni, L. et al., Nat Rev. Immunol 6 (2006): 383-393-   Gnjatic, S. et al., Proc Natl. Acad. Sci. U.S.A 100 (2003):    8862-8867-   Godkin, A. et al., Int. Immunol 9 (1997): 905-911-   Gragert, L. et al., Hum. Immunol. 74 (2013): 1313-1320-   Green, M. R. et al., Molecular Cloning, A Laboratory Manual 4th    (2012)-   Greenfield, E. A., Antibodies: A Laboratory Manual 2nd (2014)-   Gustafsson, C. et al., Trends Biotechnol. 22 (2004): 346-353-   Hwang, M. L. et al., J Immunol. 179 (2007): 5829-5838-   Jung, G. et al., Proc Natl Acad Sci USA 84 (1987): 4611-4615-   Kibbe, A. H., Handbook of Pharmaceutical Excipients rd (2000)-   Krieg, A. M., Nat Rev. Drug Discov. 5 (2006): 471-484-   Kuball, J. et al., Blood 109 (2007): 2331-2338-   Liddy, N. et al., Nat Med. 18 (2012): 980-987-   Ljunggren, H. G. et al., J Exp. Med. 162 (1985): 1745-1759-   Longenecker, B. M. et al., Ann N.Y. Acad. Sci. 690 (1993): 276-291-   Lonsdale, J., Nat. Genet. 45 (2013): 580-585-   Lukas, T. J. et al., Proc. Natl. Acad. Sci. U.S.A 78 (1981):    2791-2795-   Lundblad, R. L., Chemical Reagents for Protein Modification 3rd    (2004)-   Meziere, C. et al., J Immunol 159 (1997): 3230-3237-   Morgan, R. A. et al., Science 314 (2006): 126-129-   Mortara, L. et al., Clin Cancer Res. 12 (2006): 3435-3443-   Mueller, L. N. et al., J Proteome. Res 7 (2008): 51-61-   Mueller, L. N. et al., Proteomics. 7 (2007): 3470-3480-   Mumberg, D. et al., Proc. Natl. Acad. Sci. U.S.A 96 (1999):    8633-8638-   Pinheiro, J. et al., nlme: Linear and Nonlinear Mixed Effects Models    (http://CRAN.R-proj ect.org/packe=nlme) (2015)-   Plebanski, M. et al., Eur. J Immunol 25 (1995): 1783-1787-   Porta, C. et al., Virology 202 (1994): 949-955-   Rammensee, H. et al., Immunogenetics 50 (1999): 213-219-   Reinmuth, N. et al., Dtsch. Med. Wochenschr. 140 (2015): 329-333-   Rini, B. I. et al., Cancer 107 (2006): 67-74-   Rock, K. L. et al., Science 249 (1990): 918-921-   Rodenko, B. et al., Nat Protoc. 1 (2006): 1120-1132-   S3-Leitlinie Lungenkarzinom, 020/007, (2011)-   Saiki, R. K. et al., Science 239 (1988): 487-491-   Schmitt, T. M. et al., Hum.Gene Ther. 20 (2009): 1240-1248-   Scholten, K. B. et al., Clin Immunol. 119 (2006): 135-145-   Seeger, F. H. et al., Immunogenetics 49 (1999): 571-576-   SEER Stat facts, (2014), http://seer.cancer.gov/-   Shepherd, F. A. et al., J Clin. Oncol. 31 (2013): 2173-2181-   Sherman, F. et al., Laboratory Course Manual for Methods in Yeast    Genetics (1986)-   Singh-Jasuja, H. et al., Cancer Immunol. Immunother. 53 (2004):    187-195-   Small, E. J. et al., J Clin Oncol. 24 (2006): 3089-3094-   Sturm, M. et al., BMC. Bioinformatics. 9 (2008): 163-   Teufel, R. et al., Cell Mol Life Sci. 62 (2005): 1755-1762-   Tran, E. et al., Science 344 (2014): 641-645-   Travis, W. D. et al., J Clin. Oncol. 31 (2013): 992-1001-   Walter, S. et al., J Immunol 171 (2003): 4974-4978-   Walter, S. et al., Nat Med. 18 (2012): 1254-1261-   Willcox, B. E. et al., Protein Sci. 8 (1999): 2418-2423-   World Cancer Report, (2014)-   Zaremba, S. et al., Cancer Res. 57 (1997): 4570-4577-   Zufferey, R. et al., J Virol. 73 (1999): 2886-2892

1. A method of eliciting an immune response in a patient who has lungcancer, comprising administering to said patient a population ofactivated T cells that selectively recognize cells that present apeptide consisting of the amino acid sequence of SPRMSGLLSQT (SEQ ID NO:226).
 2. The method of claim 1, wherein the T cells are autologous tothe patient.
 3. The method of claim 1, wherein the T cells are obtainedfrom a healthy donor.
 4. The method of claim 1, wherein the T cells areobtained from tumor infiltrating lymphocytes or peripheral bloodmononuclear cells.
 5. The method of claim 1, wherein the activated Tcells are expanded in vitro.
 6. The method of claim 1, wherein thepopulation of activated T cells are administered in the form of acomposition.
 7. The method of claim 6, wherein the composition furthercomprises an adjuvant.
 8. The method of claim 7, wherein the adjuvant isselected from anti-CD40 antibody, imiquimod, resiquimod, GM-CSF,cyclophosphamide, sunitinib, bevacizumab, interferon-alpha,interferon-beta, CpG oligonucleotides and derivatives, poly-(I:C) andderivatives, RNA, sildenafil, particulate formulations with poly(lactideco-glycolide) (PLG), virosomes, interleukin (IL)-1, IL-2, IL-4, IL-7,IL-12, IL-13, IL-15, IL-21, and IL-23.
 9. The method of claim 1, whereinthe activated T cells are cytotoxic T cells produced by contacting Tcells with an antigen presenting cell that presents the peptide in acomplex with an MHC class I molecule on the surface of the antigenpresenting cell, for a period of time sufficient to activate said Tcell.
 10. The method of claim 9, wherein the antigen presenting cell isinfected with a recombinant virus expressing the peptide.
 11. The methodof claim 10, wherein the antigen presenting cell is a dendritic cell ora macrophage.
 12. The method of claim 5, wherein the expansion is in thepresence of an anti-CD28 antibody and IL-12.
 13. The method of claim 1,wherein the population of activated T cells comprises CD8-positivecells.
 14. The method of claim 9, wherein the contacting is in vitro.15. The method of claim 1, wherein the cells present the peptide at alevel at least 1.2-fold of that present in normal tissue.
 16. The methodof claim 1, wherein the immune response is capable of killing cancercells that present a peptide consisting of the amino acid sequence ofSPRMSGLLSQT (SEQ ID NO: 226).
 17. A method of eliciting an immuneresponse in a patient who has lung cancer, comprising administering tosaid patient a composition comprising a peptide in the form of apharmaceutically acceptable salt, wherein said peptide consisting of theamino acid sequence of SPRMSGLLSQT (SEQ ID NO: 226), thereby inducing aT-cell response to the lung cancer.
 18. The method of claim 17, whereinthe T cell response is a cytotoxic T cell response.
 19. The method ofclaim 17, wherein the adjuvant is selected from anti-CD40 antibody,imiquimod, resiquimod, GM-CSF, cyclophosphamide, sunitinib, bevacizumab,interferon-alpha, interferon-beta, CpG oligonucleotides and derivatives,poly-(I:C) and derivatives, RNA, sildenafil, particulate formulationswith poly(lactide co-glycolide) (PLG), virosomes, interleukin (IL)-1,IL-2, IL-4, IL-7, IL-12, IL-13, IL-15, IL-21, and IL-23.
 20. The methodof claim 17, wherein the immune response is capable of killing cancercells that present a peptide consisting of the amino acid sequence ofSPRMSGLLSQT (SEQ ID NO: 226).